Anti-infective agents against intracellular pathogens

ABSTRACT

A new class of phosphoinositide-dependent kinase-1 (PDK-1) inhibitors of Formula I: 
                         
wherein X wherein X is —CF 3 , Ar is selected from
 
                         
and R is selected from
 
                         
where
     R′ is L-Lys, D-Lys, β-Ala, L-Lue, L-Ile, Phe, SO 2 CH 2 CH 2 NH 2 , SO 2 NH 2 , Asn, Glu or Gyl, and   R″ is methyl, ethyl, allyl, CH 2 CH 2 OH, CH 2 CN, CH 2 CH 2 CN, CH 2 CONH 2 ,

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on U.S. Provisional application Ser. No.60/951,672, filed Jul. 24, 2007, as well as U.S. Provisional applicationSer. No. 60/952,158, filed Jul. 26, 2007, the priorities for which arehereby claimed and the disclosures of which are hereby incorporatedherein by reference in their entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made, at least in part, with government support underNational Institutes of Health Grant CA94829 and Army Grant DAMD17-02-1-0117. The government may have certain rights in this invention.

BACKGROUND

Francisella tularensis is a facultative, intracellular, Gram-negativebacterium that is the causative agent of tularemia, a severe andpotentially lethal zoonotic disease. This NIAID Category A pathogen hasbeen recognized for several decades as a potential threat to publichealth as a bioweapon for a number of reasons. These include its abilityto infect via multiple routes, the very low infectious dose required tocause serious disease, the acquisition by aerosol exposure ofrespiratory tularemia, the most debilitating and lethal form of thedisease, and the ease with which aerosolized organisms could be widelydisseminated. In light of recent concerns about bioterrorism, thedevelopment of new therapies to defend against the use of F. tularensisas a biological weapon is a priority.

Autophagy is a mechanism of cellular homeostasis in which cytoplasmicmaterial is sequestered in characteristic vacuoles called autophagosomesand then delivered to lysosomes for degradation. This evolutionarilyconserved process provides for the recycling of long-lived cytosolicproteins to fulfill cellular needs for energy and survival in responseto environmental stress or nutrient deprivation, and for the removal ofexcess or damaged organelles which may serve to protect cells fromapoptosis. Recent studies have established a role for autophagy incellular defense against intracellular pathogens including bacteria,such as Mycobacterium tuberculosis, Streptococcus pyogenes, Shigellaspp. and Salmonella typhimurium, as well as viruses and protozoa. Theexecution of autophagy is regulated by upstream signal transductionsystems that are influenced by largely physiological factors such asnutrient status, growth factors/cytokines, and hypoxia. Thepharmacological induction of autophagy represents an intriguing andunexploited therapeutic strategy in which this effector of innateimmunity would be triggered or amplified to defend against intracellularpathogens.

OSU-03012 (formula XV), a PDK-1/Akt signaling inhibitor, is one of manydistinct classes of molecularly targeted agents developed by theinventors. OSU-03012 was derived through structure-based optimization ofthe COX-2 inhibitor, celecoxib, with regard to PDK-1 activity. Based inpart on the novelty of its molecular target and its importance in cancercell survival, the compound entered into preclinical evaluation throughthe NCI RAID program. Investigation of OSU-03012 revealed that itinduces autophagy with a sub-/μM IC50. This point is noteworthy for tworeasons. First, concentrations in this range are clearly attainable inthe in vivo preclinical and clinical settings. Second, this IC₅₀ forautophagy induction is quite a bit lower than that for inhibition ofPDK-1 activity (˜5 μM), which suggests a mechanism distinct frominhibition of PDK-1/Akt signaling.

SUMMARY OF THE INVENTION

Provided are compounds of Formula I that induce autophagy and/or defenda host against intracellular pathogens:

wherein X is selected from alkyl and haloalkyl; Ar is selected from thegroup consisting of phenyl, biphenyl, naphthyl, anthryl, phenanthryl,and fluorenyl; R is selected from the group consisting of —CN, —CH₂CN,—CH₂CH₂CN, —CH₂CH₂CH₂CN—CONH₂

Formula I also includes pharmaceutically acceptable salts thereof,metabolism products, and prodrugs thereof. Also, provided are compoundsof formulae II-XIV that induce autophagy and/or defend a host againstintracellular pathogens. Formula II-XIV also includes pharmaceuticallyacceptable salts thereof, metabolism products, and prodrugs thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows dose-dependent effects of compound 34/70 (left panels) andcompound 35/71 (right panels) on cell viability of PC-3 cells and on thecell growth in nine representative human tumor cell lines.

FIG. 2 shows the synthesis of compounds 25-36 (61-72) using the1,1,1-trifluoro-4-hydroxy-4-phenanthren-2-yl-but-3-en-2-one as a commonprecursor.

FIG. 3 shows in panel A time-dependent and in panel B dose-dependenteffects of OSU-03012 as assessed in intracellular survival (i.e.gentamicin protection) assays; and panel C shows the effect of OSU-03012on extracellular bacterial growth in liquid medium.

FIG. 4 shows in panel A that 3-methyladenine (3MA) completely blockedthe ability of OSU-03012 to reduce intracellular bacterial survival; andpanel B shows that rapamycin, an established inducer of autophagy, isalso capable of suppressing survival of S. typhimurium in murinemacrophages.

FIG. 5 shows in panel A that the human breast cancer cell lines, MCF-7and MDA-MB-231, differ in their expression levels of beclin-1, a tumorsuppressor that is critical for the execution of autophagy; panel Bshows that intracellular bacterial survival in OSU-03012-treated S.typhimurium-infected breast cancer cells also reflects their respectivebeclin-1 status; and panel C shows that the ability of OSU-03012 toinduce autophagy in these cell lines, as determined by the intensity ofimmunostaining for endogenous LC3, a marker of mammalian autophagosomes,correlates with their respective beclin-1 expression levels.

FIG. 6 shows that OSU-03012 reduced intracellular survival of F.novicida.

DETAILED DESCRIPTION

Due to cell membrane barriers, antibiotic treatment is ineffectiveagainst intracellular bacteria as most antibiotics are unable topenetrate the cell membrane or will be excluded by host cells. Even ifan antibiotic can enter cells, intracellular bacterial growth mightcause a transient antibiotic-resistance, causing a need of higher dosesof antibiotics kill the intracellular bacteria. On the other hand, hostcells developed innate immunity that employs different strategies todefend against intracellular pathogens. Given the importance ofautophagy as an immune defense mechanism against intracellularpathogens, the ability of OSU-03012 and derivatives to eliminateintracellular bacteria in macrophages was tested. OSU-03012 andderivatives (Formulae I-XV) promote intracellular bacterial clearance byinducing autophagy in the host macrophage at physiologically attainableconcentrations. Thus, OSU-03012 and derivatives are anti-infectiveagents for the treatment of diseases caused by intracellular pathogens,including, but not limited to, Mycobacterium tuberculosis(tuberculosis), Francisella tularensis (pulmonary tularemia), Group AStreptococcus pyogenes, Rickettsiae spp., and Salmonella typhimurium.OSU-03012 and derivatives can also be used as autophagy-inducing agentsto treat viral infections and neurodegenerative diseases.

These small-molecule autophagy-inducing agents (Formulae I-XV) activatean innate defense mechanism in macrophage to eradicate bacterial hidinginside host cells. In addition, these agents can be used in theprevention of septic shock, which occurs when high doses of antibioticsare used, by reducing the release of bacterial endotoxins.

The compounds described herein can be shown in the general Formula I:

wherein X is selected from alkyl and haloalkyl; Ar is selected from thegroup consisting of phenyl, biphenyl, naphthyl, anthryl, phenanthryl,and fluorenyl; R is selected from the group consisting of —CN, —CH₂CN,—CH₂CH₂CN, —CH₂CH₂CH₂CN, —CONH₂,

Stated otherwise, R is selected from nitrile, acetonitrile,ethylnitrile, propylnitrile, carboxyamide, amidine, pyrazole, oxime,hydrazone, acetamidine, acetamide, guanidine, and urea. Formula I alsoincludes pharmaceutically acceptable salts thereof, metabolism products,and prodrugs thereof.

In some embodiments, X is C₁ to C₄ haloalkyl. In some embodiments, X isCF₃. In some embodiments, Ar may be substituted at any substitutableposition with one or more radicals, such as, but not limited to halo,C₁-C₄ alkyl, C₁-C₄ haloalkyl, azido, C₁-C₄ azidoalkyl, aryl, alkylaryl,haloaryl, haloalkylaryl, and combinations thereof. In some embodiments,Ar is selected from 2-naphthyl, 4-biphenyl, 9-anthryl, 2-fluorenyl,4-azidophenyl, 4-azidomethylphenyl, 4-(2-azidoethyl)phenyl,4-(3-azidopropyl)phenyl, 4-(4-azidobutyl)phenyl,4-(4-azidophenyl)phenyl, 4-(4-azidomethylphenyl)phenyl, 4-methylphenyl,4-ethylphenyl, 4-propylphenyl, 4-butylphenyl, 4-(2-bromoethyl)phenyl,4-(3-bromopropyl)phenyl, 4-(4-bromobutyl)phenyl,4-(trifluoromethyl)phenyl, 4-(4-methylphenyl)phenyl,4-(4-bromomethylphenyl)phenyl, 4-(4-butylphenyl)phenyl,4-(4-tert-butylphenyl)phenyl, 2-chlorophenyl, 4-chlorophenyl,2,4-dichlorophenyl, 3,4-dichlorophenyl, 2,5-dichlorophenyl,2,4-dimethylphenyl, 2,5-dimethylphenyl, 3,4-dimethylphenyl,3,5-dimethylphenyl, 4-(4-chlorophenyl)phenyl,4-(3,5-dichlorophenyl)phenyl, 4-(2,3-dichlorophenyl)phenyl,4-(3,5-dimethylphenyl)phenyl, 4-(2,4,5-trichlorophenyl)phenyl,4-(4-trifluoromethylphenyl)phenyl, 2-phenanthrenyl, 3-indolyl,2-pyrrolyl, and 4-(benzyl)phenyl. In some embodiments, Ar is selectedfrom 4-(2-bromoethyl)phenyl, 4-(3-bromopropyl)phenyl,4-(2-azidoethyl)phenyl; 4-(3-azidopropyl)phenyl, 4-butylphenyl,4-t-butylphenyl, 2-naphthalenyl, 3-indolyl, 4-biphenylyl,4′-chloro[1,1′-biphenyl]-4-yl, 3′,5′-dichloro[1,1′-biphenyl]-4-yl,2′,3′-dichloro[1,1′-biphenyl]-4-yl, 4′-methyl[1,1′-biphenyl]-4-yl,4′-trifluoromethyl[1,1′-biphenyl]-4-yl,4′-bromomethyl[1,1′-biphenyl]-4-yl, 3′,5′-dimethyl[1,1′-biphenyl]-4-yl,4′-butyl[1,1′-biphenyl]-4-yl, 4′-tert-butyl[1,1′-biphenyl]-4-yl,4-(phenylmethyl)phenyl, 9H-fluoren-2-yl, 9-anthracenyl, 2-phenanthrenyl,9-phenanthrenyl. In some embodiments, Ar is 2-phenanthrenyl. In someembodiments, R is selected from aminoacetamide and guanidine.

Another embodiment described herein is that of Formula II:

wherein X is selected from alkyl and haloalkyl; R is selected —CN,—CH₂CN, —CH₂CH₂CN, —CH₂CH₂CH₂CN —CONH₂

or stated otherwise, R is selected from nitrile, acetonitrile,ethylnitrile, propylnitrile, carboxyamide, amidine, pyrazole, oxime,hydrazone, acetamidine, acetamide, guanidine, and urea. In someembodiments, X is C₁ to C₄ haloalkyl, and in some embodiments, X is CF₃.In some embodiments, R is aminoacetamide or guanidine. Formula III alsoincludes pharmaceutically acceptable salts thereof, metabolism products,and prodrugs thereof.

Another embodiment described herein is that of Formula III:

wherein R is selected from the group consisting of —CN, —CH₂CN,—CH₂CH₂CN, —CH₂CH₂CH₂CN —CONH₂

or stated otherwise, R is selected from nitrile, acetonitrile,ethylnitrile, propylnitrile, carboxyamide, amidine, pyrazole, oxime,hydrazone, acetamidine, acetamide, guanidine, and urea. In someembodiments, R is aminoacetamide or guanidine. Formula II also includespharmaceutically acceptable salts thereof, metabolism products, andprodrugs thereof.

Some additional compounds of Formula III include the following groupsfor R:

In another embodiment, the compounds are that of Formula IV or V:

In other embodiments, the compounds are that of following FormulasVI-XIV:

In still another embodiment, the compounds are that of following FormulaXIII:

XIII

F4ME R″ = Me MW 530.51 F4E R″ = Et MW 544.54 F4HE R″ = CH2CH2OH MW560.53 F4ETFM R″ = CH2CH2CF3 MW 612.53 F4ACN R″ = CH2CN MW 555.52 F4PCNR″ = CH2CH2CN MW 569.54 F4AP R″ = —COCH2CH2

MW 573.53 F4AMO R″ = —COCH2CH2

MW 574.52 F4AMP R″ = —COCH2CH2

MW 587.56 F4AANT R″ = —CH2CH2CONH—

MW 632.54 F4AAT R″ = —CH2CH2CONH—

MW 602.55 F4AA R″ = CH2CONH2 F4PA R″ = CH2CH2CONH2

In a still another embodiment, the compound has the following FormulaXIV:

XIV

N4NBS R″ =

MW 657.66 N4BPS R″ =

MW 688.72 N4BBS R″ =

MW 691.56 N4MBS R″ =

MW 626.69 N4ME R″ = Me MW 486.53 N4E R″ = Et MW 500.56 N4ALL R″ = AllylMW 512.57 N4HE R″ = CH2CH2OH MW 516.56 N4ACN R″ = CH2CN MW 511.54 N4PCNR″ = CH2CH2CN MW 525.57 N4ETFM R″ = CH2CH2CF3 MW 568.56 N4AA R″ =CH2CONH2

In a still another embodiment, the compound has the following FormulaXV:

Generally, compounds of Formulas VI-XV (except for those formula IX) canbe regarded as corresponding to formula I

in which X is —CF₃, Ar is selected from

and R is selected from

-   R′ is L-Lys, D-Lys, β-Ala, L-Lue, L-Ile, Phe, SO₂CH₂CH₂NH₂, SO₂NH₂,    Asn, Glu or Gyl, and-   R″ is methyl, ethyl, allyl, CH₂CH₂OH, CH₂CN, CH₂CH₂CN, CH₂CONH₂,

Provided also are methods of using the compounds of formulae I-XV toinduce apoptosis in undesirable proliferating cells in subjects in needof such treatment. Also provided are methods of using compounds offormulae I-XV to induce autophagy in cells infected with intracellularpathogens in subjects in need of such treatment. The methods involvetreating the subject in need of such treatment with a therapeuticallyeffective amount of a compound of formulae I-XV or derivative,metabolites, or pharmaceutically acceptable salts thereof.

The compounds and methods described herein are useful for, but notlimited to treating, inhibiting, or delaying the onset of cancers. Thecompounds and methods are also useful in the treatment of intracellularinfections. The compounds and methods are also useful in the treatmentof precancers and other incidents of undesirable cell proliferation. Thecompounds of formula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII,XIII, XIV, or XV, or combinations thereof, are administered to a subjectthat has been diagnosed with or is at risk of developing a disordercharacterized by undesirable cell proliferation. The compounds andmethods are useful for treating cancers including, but not limited to,leukemia, melanoma, non-small cell lung cancer, colon cancer, cancers ofthe central nervous system, ovarian cancer, breast cancer, kidneycancer, and prostate cancer. Furthermore, they are useful in the slowingthe growth of these cancers in individuals with precancers, as well asindividuals prone to or having a genetic predisposition to thesedisorders.

The compounds are useful in methods of inducing apoptosis in unwantedrapidly proliferating cells, the method comprising introducing atherapeutically effective amount of a compound of formula I, II, III,IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, or XV, or combinationsthereof, to the unwanted rapidly proliferating cells. In accordance withthis method, the unwanted rapidly proliferating cells may be cancercells. The cancer cells may be selected from the group consisting ofleukemia, melanoma, non-small cell lung cancer, colon cancer, cancers ofthe central nervous system, ovarian cancer, breast cancer, kidneycancer, and prostate cancer.

The compounds are further useful for preventing restenosis in a subjectwho has undergone an angioplasty or stent procedure comprisingadministering a therapeutically effective amount of a compound offormula I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, XIV, orXV, or combinations thereof, or pharmaceutically acceptable salts and/ormetabolites thereof to the subject who has undergone an angioplasty orstent procedure. XV, or combinations thereof, or pharmaceuticallyacceptable salts and/or metabolites thereof to the subject who hasundergone an angioplasty or stent procedure.

The following terms used herein include, but are not limited to thefollowing definitions:

The term “PDK-1/Akt signaling inhibitor” signifies that a specificcompound or combination of compounds is capable of disrupting thePDK-1/Akt signaling pathway, as measured versus a blank, regardless ofwhether in vivo or in vitro. One method is set forth in the examplesbelow, though other methods now known or later developed may also beused.

The term “treatment” as used herein, encompasses the administrationand/or application of one or more compounds described herein, to asubject, for the purpose of providing prevention of or management of,and/or remedy for a condition. “Treatment” for the purposes of thisdisclosure, may, but does not have to, provide a cure; rather,“treatment” may be in the form of management of the condition. When thecompounds described herein are used to treat unwanted proliferatingcells, including cancers, “treatment” includes partial or totaldestruction of the undesirable proliferating cells with minimaldestructive effects on normal cells. A desired mechanism of treatment ofunwanted rapidly proliferating cells, including cancer cells, at thecellular level is apoptosis.

The term “prevention” as used herein includes either preventing orslowing the onset of a clinically evident unwanted cell proliferationaltogether or preventing or slowing the onset of a preclinically evidentstage of unwanted rapid cell proliferation in individuals at risk. Alsointended to be encompassed by this definition is the prevention orslowing of metastasis of malignant cells or to arrest or reverse theprogression of malignant cells. This includes prophylactic treatment ofthose at risk of developing precancers and cancers. Also encompassed bythis definition is the prevention or slowing of restenosis in subjectsthat have undergone angioplasty or a stent procedure.

The terms “therapeutically effective” and “pharmacologically effective”are intended to qualify the amount of each agent which will achieve thegoal of improvement in disease severity and the frequency of incidenceover treatment with the compounds described herein. Therapeuticallyeffective or pharmacologically effective amounts may readily bedetermined by those skilled in the art.

The term “subject” for purposes of treatment includes any human oranimal subject who has been diagnosed with, has symptoms of, or is atrisk of developing a disorder characterized by unwanted, rapid cellproliferation. Such disorders include, but are not limited to cancersand precancers. For methods of prevention the subject is any human oranimal subject. To illustrate, for purposes of prevention, a subject maybe a human subject who is at risk of or is genetically predisposed toobtaining a disorder characterized by unwanted, rapid cellproliferation, such as cancer. The subject may be at risk due toexposure to carcinogenic agents, being genetically predisposed todisorders characterized by unwanted, rapid cell proliferation, and soon. Besides being useful for human treatment, the compounds describedherein are also useful for veterinary treatment of mammals, includingcompanion animals and farm animals, such as, but not limited to dogs,cats, horses, cows, sheep, and pigs.

Dosage and Administration

Preliminary animal studies have shown that these compounds can be orallyabsorbed, can generate average serum concentrations several-fold higherthan total growth inhibition (TGI), and more importantly, incur littletoxicity to the animals after daily oral administration for one month(data not shown)

The compounds of the present invention can be formulated into suitablepharmaceutical preparations such as tablets, capsules, or elixirs fororal administration or in sterile solutions or suspensions forparenteral administration. The therapeutic agents described herein canbe formulated into pharmaceutical compositions using techniques andprocedures well known in the art.

The PDK-1/Akt signaling inhibitor described herein is compounded with aphysiologically acceptable vehicle, carrier, excipient, binder,preservative, stabilizer, flavor, etc., in a unit dosage form as calledfor by accepted pharmaceutical practice. The amount of active substancein those compositions or preparations is such that a suitable dosage inthe range indicated is obtained. In some embodiments, the dosage may bebetween 0.1 to 1000 mg of the PDK-1/Aft signaling inhibitor. In someembodiments, the compositions can be formulated in a unit dosage form,each dosage containing from 1 to 500 mg. In other embodiments, thedosage may be from 10 to 100 mg of the active ingredient. The term “unitdosage from” refers to physically discrete units suitable as unitarydosages for human subjects and other mammals, each unit containing apredetermined quantity of active material calculated to produce thedesired therapeutic effect, in association with a suitablepharmaceutical excipient. The dosage may depend on many factors, such asthe age and size of the subject, the condition being treated, theseverity of the condition, and other factors known to those skilled inthe art. Taking those factors into account, dosages can be determined bythose skilled in the art.

To prepare compositions, one or more of the therapeutic agents employedin the methods of the invention are mixed with a suitablepharmaceutically acceptable carrier. Upon mixing or addition of thetherapeutic agent(s), the resulting mixture may be a solution,suspension, emulsion, or the like. These may be prepared according tomethods known to those skilled in the art. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forinducing apoptosis in undesired cells, such as cancer cells, and may beempirically determined.

Pharmaceutical carriers or vehicles suitable for administration of thepresent therapeutic agents include any such carriers suitable for theparticular mode of administration. In addition, the active materials canalso be mixed with other active materials that do not impair the desiredaction, or with materials that supplement the desired action, or haveanother action. The present therapeutic agents may be formulated as thesole pharmaceutically active ingredient in the composition or may becombined with other active ingredients. Derivatives of the presenttherapeutic agents, such as salts or prodrugs, may also be used informulating effective pharmaceutical compositions.

The present therapeutic agents may be prepared with carriers thatprotect them against rapid elimination from the body, such astime-release formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems. The active compound can be included in thepharmaceutically acceptable carrier in an amount sufficient to exert atherapeutically useful effect in the absence of undesirable side effectson the patient treated.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

Oral compositions will generally include an inert diluent or an ediblecarrier and may be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, the activecompound or compounds can be incorporated with excipients and used inthe form of tablets, capsules, or troches. Pharmaceutically compatiblebinding agents and adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches, and the like can contain any ofthe following ingredients or compounds of a similar nature: a bindersuch as, but not limited to, gum tragacanth, acacia, corn starch, orgelatin; an excipient such as microcrystalline cellulose, starch, orlactose; a disintegrating agent such as, but not limited to, alginicacid and corn starch; a lubricant such as, but not limited to, magnesiumstearate; a glidant, such as, but not limited to, colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; and aflavoring agent such as peppermint, methyl salicylate, or fruitflavoring.

The compounds of Formulae I-XV may trigger cell death by a number ofdifferent mechanisms, however, in most embodiments, the compounds ofFormulae I-XV are able to induce apoptosis in unwanted, proliferativecells. The term “apoptosis” refers to the process of programmed celldeath. In every person hundreds of thousands of old or damaged cells dieeach day by the process of apoptosis and are replaced in the ebb andflow of maintaining a constant number of living cells in the body. Oldand damaged cells die in response to a signal triggered on the cellsurface for the targeted cell to self destruct. Apoptosis isdistinguished from other mechanisms of cell death, such as necrosis,which results in inflammation including swelling, redness, pain andtenderness. Apoptosis does not stimulate such reactions. In apoptosis,the cells shrivel up, break into pieces and the contents are quietlyremoved by methods that do not induce inflammation. For these reasons,it is highly desirable to induce apoptosis, rather than necrosis, inrapidly proliferating cells, such as cancer cells. However, mutations insome cancer cells confer resistance of these cells to apoptosis. Thecompounds of Formulae I-XV have been found to induce apoptosis even incancer cells which, because of mutations, are otherwise resistant toapoptosis. Apoptosis can be distinguished from other treatmentmechanisms by methods such as microscopy, which are known in the art.

The terms “proliferative cells,” “proliferating cells,” “rapidlyproliferating cells,” “undesirable proliferating cells,” “undesirablerapidly proliferating cells,” “unwanted rapidly proliferating cells,”and the like, refer to cancer cells, pre-cancer cells, and otherunwanted, rapidly dividing cells in a subject.

Materials and Methods

Materials4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamidewas extracted from capsules obtained from Amerisource Health (Malvern,Pa.) with ethyl acetate followed by recrystallization from a mixture ofethyl acetate and hexane. The Cell Death Detection ELISA kit waspurchased from Roche Diagnostics (Mannheim, Germany). Rabbit polyclonalantibodies against Akt and phospho-473Ser Akt were obtained from CellSignaling Technologies (Beverly, Mass.). Mouse monoclonalanti-poly(ADPribose) polymerase (PARP) antibody was provided byPharmingen (Sari Diego, Calif.). The PDK-1 kinase assay kit waspurchased from Upstate (Lake Placid, N.Y.). Other chemical andbiochemicals were obtained from Sigma-Aldrich (St. Louis, Mo.) unlessotherwise mentioned. Nuclear magnetic resonance spectra (¹H NMR) weremeasured on Bruker 250 MHz. Chemical shifts (δ) are reported in partsper million (ppm) relative to TMS peak with CDCl₃ as solvent unlessotherwise mentioned. High-resolution electrospray ionization massspectrometry analyses were performed with a 3-Tesla Finnigan FTMS-2000Fourier Transform mass spectrometer.

Synthesis of Chemicals

The compounds listed in Table 1 were prepared and tested as indicatedbelow. The chemical names, proton nuclear magnetic resonance (¹H NMR)and high-resolution mass spectrometry (HRMS) data are summarized below.The procedures used to synthesize compounds 1-36 are described in theExamples, below.

TABLE 1 Nomenclatures, 1 H NMR (proton nuclear magnetic resonance), andHRMS (high resolution mass spectrometry) characterizations of compounds1-36. Compound Description 14-[5-(4-(2-bromoethyl)phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ3.16 (t, J = 6.4, 2.0 Hz, 2H), .3.60 (t, J = 6.4, 2.0 Hz, 2 H), 4.90 (s, 2 H), 6.75 (s, 1 H), 7.13(d, J = 8.0 Hz, 2 H), 7.20 (d, J = 8.0 Hz, 2 H), 7.47 (d, J = 8.5 Hz, 2H), 7.91 (d, J = 8.5 Hz, 2 H) C₁₈H₁₅BrF₃N₃O₂S: HRMS (M + Na⁺):theoretical mass, 495.9913; actual mass, 495.9943 24-[5-(4-(3-bromopropyl)phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ 2.16 (m, 2 H), 2.81 (t, J =7.1 Hz, 2 H), 3.41 (t, J = 6.4 Hz, 2 H), .5.08 (s, 2 H), 6.76 (s, 1 H),7.15 (d, J = 8.2 Hz, 2 H), 7.25 (d, J = 8.2 Hz, 2 H), 7.47 (d, J = 8.5Hz, 2 H), 7.90 (d, J = 8.5 Hz, 2 H) C₁₉H₁₇BrF₃N₃O₂S; HRMS (M + Na⁺):theoretical mass, 510.0069; actual mass, 510.0042 34-[5-(4-(2-azidoethyl)phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ 2.90 (t, J = 6.8 Hz, 2 H),3.51 (t, J = 6.8 Hz, 2 H), .5.49 (s, 2 H), 6.76 (s, 1 H), 7.17 (d, J =8.3 Hz, 2 H), 7.24 (d, J = 8.3 Hz, 2 H), 7.42 (d, J = 8.7 Hz, 2 H), 7.85(d, J = 8.7, 2.0 Hz, 2 H) C₁₈H₁₅F₃N₆O₂S; HRMS (M + Na⁺): theoreticalmass, 459.0821; actual mass, 459.0817 44-[5-(4-(3-azidopropyl)phenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ 1.83 (m, 2 H), 2.64 (t, J =7.5 Hz, 2 H), 3.20 (t, J = 7.5 Hz, 2 H), .5.31 (br s, 2 H), 6.67 (s, 1H), 7.07 (m, 4 H), 7.35 (dd, J = 7.5, 2.0 Hz, 2 H), 7.79 (d, J = 7.5,2.0 Hz, 2 H) C₁₉H₁₇F₃N₆O₂S; HRMS (M + Na⁺): theoretical mass, 473.0978;actual mass, 473.0946 5 4-[5-(4-butylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ 0.93 (t, J = 7.2 Hz, 3 H),1.36 (m, 2 H), 1.64 (m, 2 H), 2.63 (t, J = 7.6 Hz, 2 H), 5.54 *sm 2 H),6.76 (s, 1 H), 7.15 (d, J = 8.3 Hz, 2 H), 7.20 (d, J = 8.3 Hz, 2 H),7.45 (dt, J = 8.8, 2.0 Hz, 2 H), 7.88 (dt, J = 8.8, 2.0 Hz, 2 H)C₂₀H₂₀F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass, 446.1120; actual mass,446.1149 6 4-[5-(4-t-butylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ 1.33 (s, 9 H), 4.90 (s, 2 H),6.53 (s, 1 H), 7.32 (dd, J = 9.7 Hz, 4 H), 7.42 (d, J = 8.8 Hz, 2 H),8.02(d, J = 8.8 Hz, 2 H) C₂₀H₂₀F₃N₃O₂S; HRMS (M + Na⁺): theoreticalmass, 446.1120; actual mass, 446.1118 74-[5-(2-naphthalenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide 1 H-NMR δ 5.47 (s, 2 H), 6.89 (s, 1H), 7.18 (dd, J = 8.6, 1.6 Hz, 1 H), 7.42 (bd, J = 8.6 Hz, 2 H), 7.51-7.55 (m, 2 H), 7.78-7.83 (m, 6 H) C₂₀H₁₄F₃N₃O₂S; HRMS (M + Na⁺):theoretical mass, 440.0651; actual mass, 440.0657 84-[5-(3-indolyl)-3(trifluoromethyl)-1 H-pyrazol-1-yl]benzenesulfonamide¹H-NMR δ(acetone-d₆)6.69 (br s, 1 H), 7.03-7.08 (m, 2 H), 7.19 (t, J =7.2 Hz, 1 H), 7.40 (d, J = 7.8 Hz, 1 H), 7.50 (d, J = 7.8 Hz, 1 H), 7.67(d, J = 8.7 Hz, 2 H), 7.92 (d, J = 8.7 Hz, 2 H) C₁₈H₁₃F₃N₄O₂S; HRMS (M +Na⁺): theoretical mass, 429.0603; actual mass, 429.0606 94-[5-4-biphenylyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzensulfonamide ¹H-NMR δ 4.81 (s, 2 H), 6.75 (s, 1 H),7.23 (d, J = 8.5 Hz, 2 H), 7.34-7.56 (m, 5 H), 7.56 (m, 4 H), 7.86 (d, J= 8.5 Hz, 2 H) C₂₂H₁₆F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass,466.0807; actual mass, 466.0811 104-[5-(4′-chloro[1,1′-biphenyl]-4-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ 6.42 (s, 2 H), 6.83 (s, 1 H),7.30 (d, J = 8.2 Hz, 2 H), 7.40-7.59 (m, 8 H), 7.92 (d, J = 8.2 Hz, 2 H)C₂₂H₁₅ClF₃N₃O₂S; HRMS (M + Na⁺): theoretical mass, 500.0418; actualmass, 500.0432 114-[5-(3′,5′-dichloro[1,1′-biphenyl]-4-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ 4.85 (s, 2 H), 6.82 (s, 1 H),7.30 (d, J = 8.8 Hz, 2 H), 7.36 (s, 1 H), 7.37-7.57 (m, 6 H), 7.93 (d, J= 8.8 Hz, 2 H) C₂₂H₁₄Cl₂F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass,534.0028; actual mass, 534.0016 124-[5-(2′,3′-dichloro[1,1′-biphenyl]-4-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ 4.85 (s, 2 H), 6.76 (s, 1 H),7.18-7.25 (m, 3 H), 7.35-7.49 (m, 6 H), 7.88 (d, J = 8.6 Hz, 2 H)C₂₂H₁₄Cl₂F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass, 534.0028; actualmass, 533.9999 134-[5-(2′,4′,5′-trichloro[1,1′-biphenyl]-4-yl)-3(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ¹H-NMR δ 4.86 (s, 2 H), 6.77 (s, 1 H),7.25 (dt, J = 8.6, 2.0 Hz, 2 H), 7.37 (dt, J = 8.6, s. 0 Hz, 2 H), 7.39(s, 1 H), 7.46 (dt, J = 8.8, 2.0 Hz, 2 H), 7.54 (s, 1 H), 7.88 (dt, J =8.9, 1.2 Hz, 2 H) C₂₂H₁₃Cl₃F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass,567.9638; actual mass, 567.9679 144-[5-(4′-methyl[1,1′-biphenyl]4-yl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ 2.32 (s, 3 H), 4.57 (s, 2 H), 6.72(s, 1 H), 7.18-7.21 (m, 4 H), 7.39-7.52 (m, 6 H), 7.84 (d, J = 8.9 Hz, 2H) C₂₃H₁₈F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass, 480.0964; actualmass, 480.0961 154-[5-(4′triflouromethyl[1,1′-biphenyl]-4-yl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ 5.19 (s, 2 H), 6.86 (s, 1 H), 7.36(d, J = 8.0 Hz, 2 H), 7.53 (d, J = 8.5 Hz, 2 H), 7.65 (m, 6 H), 7.92 (d,J = 8.5 Hz, 2 H) C₂₃H₁₅F₆N₃O₂S; HRMS (M + Na⁺): theoretical mass,534.0681; actual mass, 534.0677 164-[5-(4′-bromomethyl[1,1′-biphenyl]-4-yl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ 3.92 (s, 2 H), 4.93 (s, 2 H), 6.66(s, 1 H), 7.03-7.26 (m, 8 H), 7.38 (d, J = 8.6 Hz, 2 H), 7.82 (d, J =8.6 Hz, 2 H) C₂₃H₁₇BrF₃N₃O₂S; HRMS (M + Na⁺): theoretical mass,558.0069; actual mass, 558.0112 174-[5-(3′,5′-dimethyl[1,1;-biphenyl]-4-yl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ 2.40 (s, 6 H), 5.38 (br s, 2 H), 6.83(s, 1 H), 7.05 (s, 1 H), 7.25 (m, 4 H), 7.50 (dd, J = 6.7, 1.7 Hz, 2 H),7.59 (dd, J = 6.7, 1.7 Hz, 2 H), 7.92 (dd, J = 6.7, 1.7 Hz, 2 H)C₂₄H₂₀F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass, 494.1120; actual mass,494.1119 18 4-[5-(4′-butyl[1,1′-biphenyl]-4-yl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ 0.96 (t, J = 7.5 Hz, 3 H), 1.41 (m, 2H), 1.66 9m, wH), 2.68 (t, J = 7.5 Hz, 2 H), 5.20 (br s, 2 H), 6.84 (s,1 H), 7.29 (dd, J = 8.2, 2.0 Hz, 4 H), 7.53 (dt, J = 8.2, 2.0 Hz, 4 H),7.62 (d, J = 8.5 Hz, 2 H), 7.93 (d, J = 8.5 Hz, 2 H) C₂₆H₂₄F₃N₃O₂S; HRMS(M + Na⁺): theoretical mass, 522.1433; actual mass, 522.1466 194-[5-(4′-tert-buty[1,1′-biphenyl]-4-yl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ 1.35 (s, 9 H), 4.87 (s, 2 H), 6.59(s, 1 H), 7.44-7.57 (m, 6 H), 7.58 (d, J = 7.5 Hz, 2 H), 7.92 (d, J =8.7 Hz, 2 H), 8.12 (d, J = 7.5 Hz, 2 H) C₂₆H₂₄F₃N₃O₂S; HRMS (M + Na⁺):theoretical mass, 522.1433; actual mass, 522.1401 204-[5-(4-(phenylmethyl)phenyl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ 3.71 (s, 2 H), 4.74 (s, 2 H), 6.52(s, 1 H), 6.91-7.11 (m, 9 H), 7.27 (d, J = 8.9 Hz, 2 H), 7.69 (d, J =8.9 Hz, 2 H) C₂₃H₁₈F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass, 480.0964;actual mass, 580.0938 21 4-[5-(9 H-fluoren-2-yl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ 3.88 (s, 2 H), 4.64 (s, 2 H), 6.68(s, 1 H), 7.26-7.38 (m, 4 H), 7.56 (d, J = 8.7 Hz, 2 H), 7.74- 7.81 (m,3 H), 7.90 (d, J = 8.7 Hz, 2 H) C₂₃H₁₆F₃N₃O₂S; HRMS (M + Na⁺):theoretical mass, 478.0807; actual mass, 478.0771 224-[5-(9-anthracenyl)-3-(trifluoromethyl)-1 H-1-yl]benzenesulfonamide¹H-NMR δ 4.63 (s, 2 H), 6.93 (s, 1 H), 7.33 (d, J = 6.8 Hz, 2 H),7.45-7.55 (m, 8 H), 8.04 (d, J = 6.8 Hz, 2 H), 8.60 (s, 1 H)C₂₄H₁₆F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass, 490.0807; actual mass490.0769 23 4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ (600 MHz) 4.89 (s, 2 H), 6.92 (s, 1H), 7.37 (d, J = 8.5, 1.4 Hz, 1 H), 7.51 (d, J = 8.6 Hz, 2 H), 7.54-7.69(m, 3 H), 7.80 (d, J = 8.8 Hz, 1 H), 7.86-7.92 (m, 4 H), 8.64 (d, J =8.4 Hz, 2 H) C₂₄H₁₆F₃N₃O₂S; HRMS (M + Na⁺): theoretical mass, 490.0807;actual mass, 490.0805 24 4-[5-(9-phenanthrenyl)-3-(trifluoromethyl)-1H-1-yl]benzenesulfonamide ¹H-NMR δ 4.76 (s, 2 H), 6.90 (s, 1 H),7.43-7.84 (m, 11 H), 8.72 (t, J = 7.8 Hz, 2 H) C₂₄H₁₆F₃N₃O₂S; HRMS (M +Na⁺): theoretical mass, 490.0807; actual mass, 490.0833 254-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1 H-1-yl]benzenecarboxamide¹H-NMR δ 5.75-6.05 (br d, 2 H), 7.0 (s, 1 H), 7.50 (dd, J = 8.5, 1.4 Hz,1 H), 7.55 (d, J = 8.5 Hz, 2 H), 7.77 (m, 3 H), 7.88 (m, 3 H), 7.90 (m,2 H), 8.72 (m, 2 H) C₂₅H₁₆F₃N₃O; HRMS (M + Na⁺): theoretical mass,454.0038; actual mass, 454.1142 264-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1 H-1-yl]benzonitrile ¹H-NMRδ 6.91 (s, 1 H), 7.46 (s, 1 H), 7.50 (d, J = 2.0 Hz, 2 H), 7.63-7.79 (m,5 H), 7.83 (d, J = 2.0 Hz, 2 H), 7.92 (m, 1 H), 8.64 (d, J = 8.4 Hz, 2H) C₂₅H₁₄F₃N₃O; HRMS (M + Na⁺): theoretical mass, 436.1032; actual mass,436.1032 27 4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-1-yl]-N-hydroxy-benzmidine ¹H-NMR δ 7.10 (s, 1 H), 7.34 (dd, J = 4.0,0.9 Hz, 1 H), 7.36 (d, J = 0.9 Hz, 1 H), 7.37 (d, J = 0.9 Hz, 1 H),7.42-7.45 (m, 3 H), 7.46 (d, J = 0.8 Hz, 1 H), 7.51-7.52 (m, 2 H), 7.53(d, J = 0.9 Hz, 1 H), 7.57 (s, 1 H), 7.89 (s, 1 H), 7.91 (s, 1 H)C₂₅H₁₇F₃N₃O; HRMS (M + Na⁺): theoretical mass, 469.1220; actual mass,469.1247 28 5-(2-phenanthrenyl)-3-(trifluoromethyl)-4-(1H-1-tetrazol-5-ylphenyl)-1 H-pyrazole ¹H-NMR δ 6.82 (s, 1 H), 7.28 (d, J= 1.8 Hz, 1 H), 7.38 (d, J = 8.7 Hz, 2 H), 7.48-7.74 (m, 5 H), 7.74 (d,J = 2.5 Hz, 2 H), 7.95 (d, J = 8.7 Hz, 2 H), 8.47 (d, J = 8.7 Hz, 2 H)C₂₅H₁₅F₃N₆; HRMS (M + Na⁺): theoretical mass, 479.1202; actual mass,479.1225 29 4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-1-pyrazol-1-yl]-benzaldehyde oxime ¹H-NMR δ 6.81 (s, 1 H), 7.27-7.30(m, 3 H), 7.47 (d, J = 8.7 Hz, 2 H), 7.52-7.57 (m, 4 H), 76.8 (d, J =8.8 Hz, 2 H), 7.75-7.79 (m, 2 H), 8.48-8.53 (m, 2 H) C₂₅H₁₆F₃N₃O; HRMS(M + Na⁺): theoretical mass, 454.1137; actual mass, 454.1106 304-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-1-pyrazol-1-yl]-benzaldehyde hydrazone ¹H-NMR δ 6.81 (s, 1 H),7.27-7.30 (m, 2 H), 7.33 (d, J = 1.8 Hz, 1 H), 7.42 (d, J = 8.6 Hz, 1H), 7.53- 7.55 (m, 2 H), 7.57-7.60 (m, 2 H), 7.68 (d, J = 8.9 Hz, 2 H),7.75 (d, J = 1.7 Hz, 1 H), 7.80 (s, 1 H), 8.48-8.55 (m, 2 H) C₂₅H₁₇F₃N₄;HRMS (M + Na⁺): theoretical mass, 453.1297; actual mass, 453.1302 314-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-1-pyrazol-1-yl]-phyenyl}-acetonitrile ¹H-NMR δ 3.77 (S, 2h), 6.93 (S,1h), 7.29-7.43 (M, 4h), 7.66-7.86 (M, 6h), 8.65 (T, J = 7.0 Hz, 3 H)C₂₆H₁₆F₃N₃; HRMS (M + Na⁺): theoretical mass, 450.1151; actual mass,450.1188 32 2-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-1-pyrazol-1-yl]-phenyl}-N-hydroxy-acetamidine ¹H-NMR δ 3.30 (s, 1 H),3.38 (s, 1 H), 6.83 (s, 1 H), 7.20-7.41 (m, 4 H), 7.59-7.89 (m, 6 H),8.55-8.60 (m, 3 H) C₂₆H₁₉F₃N₄O; HRMS (M + Na⁺): theoretical mass,461.1580; actual mass, 461.1584 335-(2-phenanthrenyl)-3-(trifluoromethyl)-4-(1H-tetrazol-5-ylmethylphenyl)-1 H-pyrazole ¹H-NMR δ 4.45 (s, 2 H), 7.15(s, 1 H), 7.42 (s, 4 H), 7.53 (d, J = 6.9 Hz, 1 H), 7.66-7.76 (m, 3 H),7.89 (d, J = 7.2 Hz, 1 H), 8.01 (m, 2 H), 8.78 (t, J = 6.9 Hz, 2 H)C₂₆H₁₇F₃N₆; HRMS (M + Na⁺): theoretical mass, 493.1335; actual mass,493.1359 34 2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}acetamide ¹H-NMR δ 3.48 (s, 2 H), 6.92 (s, 1 H),7.35 (d, J = 8.8 Hz, 2 H), 7.42 (dd, J = 8.6, 1.7 Hz, 1 H), 7.62- 7.72(m, 5 H), 7.79 (d, J = 8.8 Hz, 1 H), 7.85-7.94 (m, 2 H), 8.62 (t, J =8.5 Hz, 2 H), 9.56 (br s 1 H) C₂₆H₁₉F₃N₄O; HRMS (M + Na⁺): theoreticalmass, 483.1403; actual mass, 483.1389 354-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl-guanidine ¹H-NMR δ 6.90 (s, 1 H), 7.19 (d, J =8.7 Hz, 2 H), 7.34 (dd, J = 8.7, 2.0 Hz, 1 H), 7.39 (d, J = 8.7 Hz, 2H), 7.61-7.67 (m, 3 H), 7.79 (d, J = 9.0 Hz, 1 H), 7.84-7.91 (m, 3 H),8.62 (d, J = 8.3 Hz, 2 H), 9.95(s, 1 H) C₂₅H₁₈F₃N₅; HRMS (M + H):theoretical mass, 446.1587 (M + H); actual mass, 446.1596 (M + H) 364-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1 H-pyrazol-1-yl]-phenyl-urea¹H-NMR δ 6.98 (s, 1 H), 7.19 (dt, J = 8.9, 2.1 Hz, 2 H), 7.34-7.42 (m, 3H), 7.51-7.62 (m, 4 H), 7.70 (d, J = 9.0 Hz, 1 H), 7.81-7.85 (m, 2 H),8.59-8.64 (m, 2 H) C₂₅H₁₇F₃N₄O; HRMS (M + Na⁺): theoretical mass,469.1252; actual mass, 469.1199

Cell Culture PC-3 (p53−/−) human androgen-non-responsive prostate cancercells were purchased from the American Type Tissue Collection (Manassas,Va.). Cells were cultured in RPMI 1640 medium (Gibco, Grand Island,N.Y.) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37° C. ina humidified incubator containing 5% CO₂.

Cell viability analysis The effect of4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamideand its derivatives on PC-3 cell viability was assessed by using the MTT{[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide]}assay in six replicates. Cells were grown in 10% FBS-supplemented RPMI1640 medium in 96-well, flat bottomed plates for 24 h, and were exposedto various concentrations of compounds 1-36 dissolved in DMSO (finalconcentration ≦0.1%) in 1% serum-containing RPMI 1640 medium fordifferent time intervals. Controls received DMSO vehicle at aconcentration equal to that in drug-treated cells. The medium wasremoved, replaced by 200 μl of 0.5 mg/ml of MTT in 10% FBS-containingRPMI-1640 medium, and cells were incubated in the CO₂ incubator at 37°C. for 2 h. Supernatants were removed from the wells, and the reducedMTT dye was solubilized in 200 μL/well DMSO. Absorbance at 570 μm wasdetermined on a plate reader.

Cell proliferation PC-3 cells were seeded into six-well plates at 50,000cells/well in 10% FBS-containing RPMI 1640 medium. Following a 24 hattachment period, cells were treated in triplicate with the indicatedconcentration of compounds 1-36 or DMSO vehicle in 10% FBS-containingRPMI-1640 medium. At different time intervals, cells were harvested bytrypsinization, and numerated using a Coulter counter model Z1 D/T(Beckman Coulter, Fullerton, Calif.).

Apoptosis analysis Two methods were used to assess drug-inducedapoptotic cell death: detection of DNA fragmentation by the Cell DeathDetection ELISA kit (Roche Diagnostics. Mannheim, Germany) and Westernblot analysis of poly-(ADP-ribose)polymerase (PARP) cleavage. The ELISAwas performed according to the manufacturer's instructions, and is basedon the quantitative determination of cytoplasmic histone-associated DNAfragments in the form of mononucleosomes or oligonucleosomes generatedafter induced apoptotic death. In brief, 4×10⁵ PC-3 cells were culturedin a T-25 flask for 24 h before treatment. Cells were treated with theDMSO vehicle or the test agent at the indicated concentrations for 6-24h, collected, and cell lysates equivalent to 2×10³ PC-3 cells were usedin the ELISA. For the PARP cleavage assay, drug-treated cells werecollected 4-8 h post-treatment, washed with ice-cold PBS, andresuspended in lysis buffer containing 20 mM Tris-HCl, pH 8, 137 mMNaCl, 1 mM CaCl₂, 10% glycerol, 1% Nonidet P-40, 0.5% deoxycholate, 0.1%SDS, 100 μM 4-(2-aminoethyl)benzenesulfonyl fluoride, leupeptin at 10μg/ml, and aprotinin at 10 μg/mL. Soluble cell lysates were collectedafter centrifugation at 10,000 g for 5 min. Equivalent amounts ofproteins (60-100 μg) from each lysate were resolved in 8%SDS-polyacrylamide gels. Bands were transferred to nitrocellulosemembranes, and analyzed by immunoblotting with anti-PARP antibody.

Immunoblotting. The general procedure for the Western blot analysis ofAkt and phospho-Akt is described as follows. Cells were washed in PBS,resuspended in SDS sample buffer sonicated by an ultrasonic sonicatorfor 5 sec, and boiled for 5 min. After brief centrifugation, equivalentprotein concentrations from the soluble fractions were resolved in 10%SDS-polyacrylamide gels on a Minigel apparatus, and transferred to anitrocellulose membrane using a semi-dry transfer cell. The transblottedmembrane was washed three times with TBS containing 0.05% Tween 20(TBST). After blocking with TBST containing 5% nonfat milk for 60 min.,the membrane was incubated with the primary antibody at 1:1,000 dilutionin TBST-5% low fat milk at 4° C. for 12 h, and was then washed threetimes with TBST. The membrane was probed with goat anti-rabbit IgG-HRPconjugates (1:1,000) for 1 h at room temperature, and was washed withTBST three times. The immunoblots were visualized by enhancedchemiluminescence.

PDK-1 kinase activity This in vitro assay was performed using a PDK-1kinase assay kit (Upstate, Lake Placid, N.Y.) according to the vendor'sinstructions. This cell-free assay is based on the ability ofrecombinant PDK-1, in the presence of DMSO vehicle or the test agent, toactivate its downstream kinase serum- and glucocorticoid-regulatedkinase (SGK.) which, in turn, phosphorylates the Akt/SGK-specificpeptide substrate RPRAATF with [γ-³²]-ATP. The [³²P]-phosphorylatedpeptide substrate was then separated from the residual [γ-³²P]-ATP usingP81 phosphocellulose paper and quantitated by a scintillation counterafter three washes with 0.75% phosphoric acid. The reported valuesrepresent the means of two independent determinations.

Immunoprecipitated Akt kinase assay Akt immunoprecipitation was carriedout according to a modified, published procedure. PC-3 cells weretreated with DMSO vehicle or the test agents at the indicatedconcentrations for 2 h and then lysed at 4° C. for 1 h in buffer Acontaining 50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1 mM EDTA, 1 mMEGTA, 50 mM sodium fluoride, 10 mM sodium β-glycerophosphate. 0.1%2-mercaptoethanol, 0.1 mM phenylmethylsiilfonyl fluoride, and 1 μg/mLeach of aprotinin, pepstatin, and leupeptin. Cell lysates werecentrifuged at 10,000 g for 5 min, and the supernatant was treated withanti-Akt at 4° C. for 60 min., followed by protein G-agarose beads foradditional 60 min. The immunoprecipitate was used to analyze Akt kinaseactivity by using the Akt/SGK-specific peptide substrate RPRAATF asdescribed above. Values represented the means of two independentdeterminations.

Statistical analysis Each experiment was performed in triplicate, unlessotherwise mentioned. All experiments were carried out at least two timeson different occasions. Where appropriate, the data are presented as themean±95% confidence interval.

The structure and potency in inhibiting PDK-1 kinase activity and PC-3cell growth of 24 representative derivatives are summarized in Table 2.

TABLE 2 Structures and potency for inhibiting recombinant PDK-1 kinaseactivity and for inducing apoptotic death in PC-3 cells for 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamideand compounds 37-60 (1-24)

IC₅₀ (μM) Number Ar PDK-1 PC-3 Com- parative Com- pound

48 30 37

42 18 38

38 17 39

32 17 40

34 18 41

20  9 42

34 18 43

24 11 44

65 31 45

21 11 46

22  9 47

18 10 48

23 10 49

 9  5 50

15  8 51

18  8 52

20 11 53

17  9 54

32 15 55

32 15 56

15  8 57

16  9 58

12  7 59

 9  5 60

42 23

These compounds, except the indole derivative 44, showed improved PDK-1inhibitory and anti-proliferative activities vis-à-vis4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide. Additionally, none of these compounds displayedmeasurable COX-2 inhibitory activity (data not shown). A generalincrease in PDK-1 inhibitory activity was noted with increasingbulkiness of the aromatic ring, i.e., tricyclic aromatic rings(57-59)>substituted biphenyl (45-55)>substituted phenyl (37-42). Thesedata suggested that the aromatic system bound to a large, hydrophobicregion of the enzyme pocket. Among the 24 analogues examined, compound59 represented the optimal derivative with IC₅₀ values of 9 μM and 5 μMfor inhibiting PDK-1 activity and PC-3 cell viability, respectively, asreported in Table 2. These IC₅₀ values corresponded to a five- tosix-fold improvement over the activities of4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (48 μM and 30 μM, respectively). However, compound 60exhibited a decrease, compared with compound 59 in PDK-1 inhibitoryactivity, which might be attributable to steric hindrance imposed by anunfavorable orientation of the tricyclic aromatic ring.

There existed a correlation between PDK-1 and PC-3 growth inhibitionpotency in all compounds examined, suggesting the mechanistic relevanceof PDK-1 inhibition to the anti-proliferative effect. Overall, the IC₅₀value for inhibiting PC-3 cell proliferation was approximately one halfof that of PDK-1 inhibition. This discrepancy might arise from amechanistic synergy between PDK-1 inhibition and concomitant Aktdephosphorylation by protein phosphatase 2A (PP2A) in Aug-treated cells,resulting in augmented Akt deactivation. To examine this premise, PC-3cells were treated with different concentrations of compound 59 for 2 h,and the consequent effect on AM was assessed by two independent assays:immunoprecipitated Akt kinase activity and Akt phosphorylation status.Both assays gave consistent results.

According to the kinase assay, the IC₅₀ of compound 59 for inhibitingintracellular Akt activation was 5 μM. Neither compound 59 nor other theother compounds displayed a direct inhibitory effect onimmunoprecipitated Akt activity. Meanwhile, Western blot analysis showsthat treatment of PC-3 cells with compound 59 at 5 μM and above led tosignificant Akt dephosphorylation.

The inhibition of PDK-1/Akt signaling led to apoptotic death in PC-3cells in 1% FBS-containing RPMI 1640 medium in a dose-dependent manner,as was evidenced by DNA fragmentation and PARP cleavage. The dose ofcompound 59 required to induce 50% PC-3 cell death at 24 h was 5 μM. TheIC₅₀ values for compound 59 to induce PC-3 cell death was consistentwith that of inhibiting Akt activation in drug-treated cells.Furthermore, the effect of compound 59 on PC-3 cell proliferation wasexamined in 10% FBS-supplemented RPMI 1640 medium. Compound 59 at 1 μMshowed substantial anti-proliferative activity. Together, these dataclearly indicated the in vitro efficacy of compound 59 in PC-3 growthinhibition.

The modeling showed that compound 59 was docked into the ATP-bindingdomain that is located within a deep cleft between the two lobes ofPDK-1. Although compound 59 competed with ATP for binding, the mode ofbinding for compound 59 was found to be somewhat different from that ofATP. While the benzenesulfonamide moiety occupied the adenine-bindingmotif, the planar pyrazole moiety was perpendicular to the ribose ring.This arrangement positioned the adjacent phenanthrene ring behind thetrisphosphate-binding pocket. The phenanthrene ring formed hydrophobicinteractions with an apolar region formed by residues 88-96 encompassingpart of two adjacent β sheets joined by a glycine-rich loop.

Structures of twelve representative derivatives, their potency againstPDK-1, and their ability to cause apoptotic death in PC-3 cells aresummarized in Table 3.

TABLE 3 Structures and potency for inhibiting recombinant PDK-1 kinaseactivity and for inducing apoptotic death in PC-3 cells for compounds25-36. The general structures of these compounds is shown at top.

IC₅₀ (μM) Number R PDK-1 PC-3 61 —CONH₂ 12  7 62 —CN 45 30 63

40 25 64

52 32 65

25 14 66

16 10 67 —CH₂CN 42 25 68

15  8 69

45 27 70

 5  5 71

 2  3 72

40 24

Among these derivatives, compound 70 and 71 exhibited IC₅₀ values forPDK-1 inhibition of 5 μM and 2 μM, respectively, which represented two-and five-fold increases in potency over compound 59. Compounds 70 and 71contained side chains of 2-aminoacetamide (—NHC(O)CH₂NH₂,) and guanidine(—NHC(═NH)NH₂), respectively. Like compound 59, they exhibited noappreciable direct inhibition on immunoprecipitated Akt kinase activity,nor was any measurable COX-2 inhibitory activity detected atconcentrations up to 50 μM. Exposure of PC-3 cells to either agent, evenat 1 μM, resulted in a substantial decrease in the phospho-Akt level.This improvement in potency reflected a strengthening of the hydrogenbonding in the protein-ligand interactions for these derivatives. Thispremise was supported by the modeled docking of compound 71 into theATP-binding site. The guanidino group of compound 71 resembled thepartial structure of ATP's purine ring, which allowed the formation ofhydrogen bonds with Ser160 and Ala162 as depicted by the docking model.

Cellular effects of PDK-1/Akt signaling inhibitors Both compound 34/70and compound 35/71 induced apoptotic death in PC-3 cells in 1%FBS-containing medium in a dose-dependent manner, as was demonstrated byDNA fragmentation and PARP cleavage. These agents exhibited higherpotency than compound 59 in apoptosis induction at concentrationsgreater than 2.5 μM. Moreover, these derivatives were submitted to theDevelopmental Therapeutic Program (DTP) at the National Cancer Institute(NCI) for screening against sixty human tumor cell lines, representingleukemia, melanoma, and cancers of the lung, colon, brain, ovary,breast, prostate, and kidney. Dose-response data of one representativecell line from each class of tumor cells after two-day exposure in 5%FBS-containing medium are shown in FIG. 1C, which include: 1, RPMI-8226leukemia cells; 2, NCI-H322M non-small cell lung cancer cells; 3, HT29colon cancer cells; 4, U251 CNS cancer cells; 5, SK-MEL-28 melanomacancer cells; 6, SK-OV-3 ovarian cancer cells; 7, RXF 393 renal cancercells; 8, PC-3 prostate cancer cells; 9, MDA-MB-231 breast cancer cells.Many of these cell lines were responsive to the growth inhibitory effectof both agents at concentrations as low as 0.1 μM

In the sixty cell line assay, three dose response parameters for eachcell line were calculated based on growth inhibition curves. Theseparameters include G150 (concentration resulting in 50% growthinhibition), TGI (concentration resulting in total growth inhibition),and LC50 (concentration resulting in a 50% reduction in the measuredprotein level at the end of drug treatment as compared to that at thebeginning). The means of these parameters among the sixty different celllines for compounds 70 and 71 after two-day treatment were as follows,respectively, G150: 1.1 and 1.2 μM; TGI: 3.2 and 2.9 μM; LC50: 24 and8.5 μM. These data clearly demonstrate the in vitro efficacy ofcompounds 70 and 71. Both agents were able to completely suppress cellgrowth in a diverse range of tumor cell lines at the 3-5 μM therapeuticrange.

In light of the conserved role of PDK-1/Akt signaling in cancer cellsurvival and proliferation, this pathway represents a therapeuticallyrelevant target for developing orally bioavailable, small-moleculeinhibitors.

In silico docking of compound 59 into the ATP-binding pocket showed thatthe molecule was anchored into the ATP binding domain, in part, throughhydrogen bonding between the sulfonamide and the amide of Ala162. Ala162has also been reported to play a key role in anchoring other ligandssuch as ATP¹⁷ and UCN-01 to PDK-1. Together, these data suggest that thesulfonamide moiety of compound 59 might be amenable to alterations foroptimizing potency.

Accordingly, replacement of the sulfonamide function with2-aminoacetamide (—NHC(O)CH₂NH₂) and guanidine [—NHC(═NH)NH₂] led tocompounds 70 and 71, respectively, both of which exhibited improvedPDK-1 inhibition with IC₅₀ values of 5 and 2 μM, respectively. Dockingof compound 71 into the ATP binding site revealed the existence of anadditional hydrogen bond between the guanidine moiety and thebackbone-oxygen of Ser160, suggesting that the enhancement in potencymight be attributable to an increase in hydrogen bonding. The effect ofthese side chains on ligand binding, however, is subtle, as illustratedby the structure-activity relationship summarized in Table 3.

The high potency of compounds 70 and 71 in PDK-1 inhibition wasreflected in their abilities to effectively block Akt activation and toinduce apoptotic cell death in PC-3 cells at low μM concentrations (FIG.1A, B). More importantly, due to the conserved role of PDK-1/Aktsignaling in cell proliferation and survival, these agents were potentin inhibiting cell growth in serum-containing medium in all 60 humantumor cell lines examined, with mean GI50 (50% cell growth inhibition)values of 1.2 μM and 1.3 μM, respectively, arid TGI (total growthinhibition) values of 3.2 μM and 2.9 μM, respectively. Our preliminaryanimal studies have shown that these compounds can be orally absorbed,can generate average serum concentrations several-fold higher than TGI,and more importantly, incur little toxicity to the animals after dailyoral administration for one month (data not shown).

General Synthetic Procedures for Compounds 37-60

All chemical reagent and organic solvents were purchased from Aldrich(St. Louis, Mo.) unless otherwise mentioned. Compounds 1-24 weresynthesized according to a two-step general procedure described inScheme 1, in which Ar represents the respective aromatic ringstructures.

Compound 59 is used here as an example to illustrate the synthesis ofthe group of compounds (Scheme 2). Other compounds followed the sameprocedures via precursors and the respective intermediates withdifferent aromatic ring structures (compounds I and II).

EXAMPLE 1

Synthesis of the1,1,1-Trifluoro-4-hydroxy-4-phenanthren-2-yl-but-3-en-2-one Precursor(step 1). To a suspension of sodium hydride (NaH; 0.13 g, 5.4 mmol) in 5mL of anhydrous tetrahydrofuran (THF) was added ethyl trifluoracetate(CF₃COOEt; 0.64 g, 4.5 mmol) under argon. After stirring at 25° C. for10 minutes, 2-acetylphenanthrene (1 g, 4.5 mmol) in 5 mL of THF wasadded dropwise to the solution. The mixture became clear and orange-huedwithin 30 minutes, and after stirring for an additional 2 hours, wasconcentrated under vacuum. The residue was suspended in water, andextracted with ethyl acetate (15 mL) twice. The organic phase wasseparated, dried over sodium sulfate, and concentrated to dryness undervacuum to give the product (yellow solid; 1.29 g, 90% yield). Theproduct was used directly without further purification.

EXAMPLE 2

Synthesis of Compound 59 (step 2). 4-Hydrazinobenzene-1-sulfonamidehydrochloride (1.1 g; 4.9 mmol) was added to a stirred solution of1,1,1,-trifluoro-4-hydroxy-4-phenanthren-2-yl-but-3-en-2-one (1.29 g,4.1 mmol) in 40 mL of ethanol. The mixture was refluxed for 12 hours,cooled to room temperature, and concentrated to dryness under vacuum.The residue was dissolved in ethyl acetate, and washed with water. Theorganic layer was dried over sodium sulfate, and concentrated undervacuum. The crude product was purified by silica gel flashchromatography to yield 59 (1.52 g, 80% yield).

EXAMPLES 3-14

Syntheses of Compounds 61-72 Compounds 61-72 (25-36) were synthesizedusing 1,1,1-trifluoro-4-hydroxy-4-phenanthren-2-yl-but-3-en-2-one,product of the aforementioned step 1, as a common precursor (Scheme 3,FIG. 2).

EXAMPLE 3

4-[5-(2-Phenanthracenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-benzenecarboxamide(61) (step 3). (4-Carbamoylphenyl)-hydrazine hydrochloride (0.92 g, 4.9mmol) was added to a stirred solution of1,1,1-trifluoro-4-hydroxy-4-phenanthren-2-yl-but-3-en-2-one (1.29 g, 4.1mmol) in 40 mL of ethanol at 25° C. The mixture was refluxed for 12hours, cooled to room temperature and concentrated to dryness undervacuum. The residue was dissolved in ethyl acetate, and washed withwater. The organic layer was dried over sodium sulfate, and concentratedunder vacuum. The crude product was purified by silica gel flashchromatography (ethyl acetate-hexane, 1:1), —yielding 61 (1 g, 60%yield).

EXAMPLE 4

4-[5-(2-Phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-benzonitrile(62) (step 4). To a stirred solution of1,1,1-trifluoro-4-hydroxy-4-phenanthren-2-yl-but-3-en-2-one (2.45 g, 7.7mmol) in 60 mL of ethanol was added 4-cyanophenylhydrazine hydrochloride(2.53 g, 15 mmol) at 25° C. The mixture was stirred under reflux for 12hours, cooled to room temperature and concentrated to dryness undervacuum. The residue was dissolved in methylene chloride, and washed withwater. The organic layer was dried over sodium sulfate, and concentratedunder vacuum. The crude product was purified by silica gel flashchromatography (ethyl-acetate-hexane, 1:4) to afford 62 (2.7 g, 85%yield).

EXAMPLE 5

4-[5-(2-Phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-N-hydroxybenzamidine(63) (step 5). Hydroxylamine hydrochloride (25 mg, 0.36 mmol) was addedto a suspension of Na metal (8.3 mg, 0.36 mmol) in methanol (3 mL). Themixture was stirred at room temperature for 10 minutes. and compound 62(1224 mg, 0.3 mmol) was added. The mixture was refluxed for 2 hours,then stirred at 25° C. for an additional 16 hours, and concentratedunder vacuum. The residue was purified by silica gel flashchromatography (ethyl acetate-hexane, 1:4 to 1:1) to give 63 (120 mg,76% yield).

EXAMPLE 6

5-(2-Phenanthrenyl)-3-(trifluoromethyl)-4-(1H-tetrazol-5-ylphenyl)-1H-pyrazole(64) (step 6). A mixture containing compound 62 (125 mg, 0.3 mmol),NH₄Cl (123.7 mg), and NaN₃ (58.5 mg, 0.9 mmol) in 5 mL of 10% HCl wasadded, and extracted with 20 mL of methylene chloride, twice. Theorganic phase was dried over sodium sulfate, and concentrated to drynessunder vacuum. The crude product was purified by silica gel flashchromatography (ethyl acetate-hexane 1:4) to give 64 (96 mg, 70% yield).

EXAMPLE 7

4-5-[-(2-Phenanthrenyl)-3-(trifluoromethyl)-1H-pyraxol-1-yl]-benzaldehydeoxime (65) (step 7). DIBAL-H (3.1 mL, 3.1 mmol, 1.0 M in hexane) wasadded dropwise to a solution of compound 62 (0.417 g, 1.1 mmol) in 5 mLTHF at −40° C. The mixture was stirred for 8 hours, poured into 5 mL of10% acetic acid, and stirred for 30 minutes. The organic layer was driedover sodium sulfate, and concentrated to dryness under vacuum. The crudeproduct was purified by silica gel flash chromatography (ethylacetate-hexane, 1:4) to give an aldehyde intermediate (141 mg, 0.34mmol) that was immediately added to a solution containing hydroxylamidehydrochloride (211 mg) and K₂CO₃ in 5 mL of ethanol. The mixture wasstirred under reflux for 16 hours. After removal of solvent, the residuewas extracted with CH₂Cl₂ and washed with water.

EXAMPLE 8

4-[5-(2-Phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-benzaldehydehydrazone (66) (step 8). Compound 66 (124 mg, 85% yield) was synthesizedin the same manner as 65 except that hydrazine monohydrate (153 mg, 3.1mmol) was used instead of hydroxylamine hydrochloride.

EXAMPLE 9

{4-[5-(2-Phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}-acetonitrile(67) (step 9). (a) Preparation of (4-Hydrazinophenyl)acetonitrilehydrochloride. A solution of sodium nitrite (3.15 g, 45.7 mmol) in water(20 mL) was added dropwise to a cooled (−15° C.), stirred suspension of4-aminobenzonitrile (5 g, 42.3 mmol) in a concentrated hydrogen chloridesolution (55 mL) at such a rate as to maintain a temperature below −10°C. After the addition was finished, the reaction mixture was quicklyfiltered to remove solids, and the filtrate was added in portions to acooled (−20° C.), stirred solution of SnCl₂.2H₂O (47.7 g, 0.21 mol) in aconcentrated hydrogen chloride solution (37 mL) at such a rate as tokeep the temperature below −10° C. After stirring the solution for anadditional 15 minutes, the solid was collected, washed with diethylether (4×25 mL), and dried to give (4-hydrazinophenyl)acetonitrilehydrochloride (5.6 g, 78%). (b) Compound 67. A mixture of(4-hydrazinophenyl)acetonitrile hydrochloride (0.32 g, 1 mmol) and1,1,1-trifluoro-4-hydroxy-4-phenanthren-2-yl-but-3-en-2-one (0.18 g, 1.1mmol) in ethanol (20 mL) was stirred under reflux for 24 hours, cooledto room temperature, concentrated to dryness under vacuum, and dissolvedin ethyl acetate. The organic layer was dried over magnesium sulfate,and concentrated to dryness under vacuum. The crude product was purifiedby silica gel column chromatography (hexane-ethyl acetate, 2:1) to givecompound 67 (0.35 g, 81% yield).

EXAMPLE 10

2-{4-[5-(2-Phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}-N-hydroxy-acetamidine(68) (step 10). A solution of compound 67 (0.43 g, 1 mmol) andhydroxyamine hydrochloride (0.075 g, 1.1 mmol) in ethanol (10 mL) wasstirred under reflux for 8 hours, and concentrated to dryness undervacuum. The residue was dissolved in water, brought to pH 8-9 byaddition of saturated NaHCO₃ solution, and extracted with ethyl acetate.The organic layer was dried over magnesium sulfate, and concentrated todryness under vacuum. The crude product was re-crystallized in diethylether-hexane to give compound 68 (0.32 g, 71% yield).

EXAMPLE 11

5-(2-Phenanthrenyl)-3-(trifluoromethyl)-4-(1H-tetrazol-5-ylmethylphenyl)-1H-pyrazole(69) (step 11). A mixture containing compound 67 (0.43 g, 1 mmol),sodium azide (0.08 g, 1.2 mmol), and triethylamine hydrochloride (0.12g, 1.2 mmol) in toluene (5 mL) was stirred at 100 C for 5 hours, cooledto room temperature, and extracted with water (10 mL). To the aqueousphase was added dropwise a 36% hydrogen chloride solution to salt outthe resulting tetrazole 69. After filtration, the solid was dried undervacuum, yielding compound 33 (0.39 g, 84% yield).

EXAMPLES 12-14

1-(4-Nitrophenyl)-5-phenyl-3-(trifluoromethyl)-1H-pyrazole (III) (step12). To a solution of1,1,1-trifluoro-4-hydroxy-4-phenanthren-2-yl-but-3-en-2-one (1.29 g, 4.1mmol) in 40 mL of ethanol was added 4-nitrophenylhydrazine hydrochloride(0.93 g, 4.9 mmol) under stirring, refluxed for 1 hour, cooled to roomtemperature, and concentrated to dryness under vacuum. The residue wasdissolved in ethyl acetate, and washed with water. The organic phase wasdried over magnesium sulfate, and concentrated to dryness under vacuum.The crude product was purified by silica gel column chromatography toafford compound III (0.88 g, 50% yield).

4-[5-(2-Phenanthrenyl)-3-(trifluoromethyl)-1H-pyrozol-1-yl]phenylamine(IV) (step 13). To a solution of compound III (0.88 g, 2 mmol) in 20 mLethanol was added platinum oxide (27 mg, 0.12 mmol), stirred under H₂ at55 psi for 12 hours, filtered to remove the catalyst, and concentratedto dryness under vacuum. The crude product was purified by silica gelchromatography to yield compound IV (0.57 g, 70% yield).

EXAMPLE 12

2-Amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}-acetamide(70) (steps 14 and 15). To a solution of t-butyloxycarbonyl(tBOC)-glycine (0.25 g, 1.4 mmol) and compound IV (0.57 g, 1.4 mmol) in10 mL of tetrahydrofuran was added1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.41 g,2.1 mmol), stirred at 25° C. for 12 hours, and concentrated to drynessunder vacuum in a rotary evaporator. The residue was suspended in water,and the product was extracted with ethyl acetate. The organic phase wasdried over magnesium sulfate, and concentrated to dryness under vacuumto give compound V (0.67 g, 85% yield). Compound V (0.67 g, 1.2 mmol)was dissolved in 8 mL of ethyl acetate containing 0.7 mL of concentratedHCl solution, stirred at room temperature for 2 hours, and concentratedto dryness under vacuum. The crude product was purified y silica gelcolumn chromatography to yield compound 70 as a white powder (0.49 g,90%).

EXAMPLE 13

4-[5-(2-Phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl-guanidine(71) (step 16). To a solution of compound IV (0.57 g, 1.4 mmol) in 7 mLof ethanol was added cyanamide (89 mg, 2.1 mmol) and 1.5 mL of 1N HCl.The mixture was refluxed for 24 hours, and concentrated to dryness undervacuum. The product was purified by silica gel column chromatography togive compound 71 as a white solid (0.25 g, 40% yield).

EXAMPLE 14

4-[5-(2-Phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl urea(72) (step 17). Into a 250 mL round bottom flask containing acetic acid(50 mL), water (12 mL), and ethanol (20 mL) was added compound IV (2.25g, 5.6 mmol), followed by sodium isocyanate (0.74 g, 11.2 mmol). Thereaction was stirred for 1.5 hours, and then neutralized with theaddition of 1 N sodium hydroxide followed by sodium hydroxide pelletsuntil the pH had changed to 7.0. The product was separated and thenwashed with 100 mL of water, dried with magnesium sulfate, and thensolvent was removed to obtain the crude product. Purification wasperformed by silica gel chromatography with (hexane-ethyl acetate, 3:2to hexane-acetone, 1:3) to afford compound 72.

EXAMPLE 15

Preparation of Additional Compounds The compounds in Table 4 wereprepared using the methods of the Examples above.

TABLE 4 Additional Compounds

Compound R Nomenclature 73

4-(5-Phenanthren-2-yl-3-trifluoromethyl-pyrazol-1-yl)-N-(4-sulfamoyl-phenyl)-benzamide 74

N-[4-(5-Phenanthren-2-yl-3-trifluoromethyl-pyrazol-1-yl)-phenyl]-4-sulfamoyl-benzamide 75

2-Guanidino-N-[4-(5-phenanthren-2-yl-3-trifluoromethyl-pyrazol-1-yl)-phenyl]-acetamide 76

2-[4-(5-Phenanthren-2-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylamino]-acetamide 77

N-{[4-(5-Phenanthren-2-yl-3-trifluoromethyl-pyrazol-1-yl)-phenylcarbamoyl]-methyl}-benzamide

EXAMPLE 16

Screening of compounds 70 and 71 against several cancer cell lines Thehuman tumor cell lines of the cancer screening panel are grown in RPMI1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For atypical screening experiment, cells are inoculated into 96 wellmicrotiter plates in 100 μL at plating densities ranging from 5,000 to40,000 cells/well depending on the doubling time of individual celllines. After cell inoculation, the microtiter plates are incubated at37° C., 5% CO₂, 95% air and 100% relative humidity for 24 h prior toaddition of experimental drugs.

After 24 h, two plates of each cell line are fixed in situ with TCA, torepresent a measurement of the cell population for each cell line at thetime of drug addition (Tz). Experimental drugs are solubilized indimethyl sulfoxide at 400-fold the desired final maximum testconcentration and stored frozen prior to use. At the time of drugaddition, an aliquot of frozen concentrate is thawed and diluted totwice the desired final maximum test concentration with complete mediumcontaining 50 μg/ml gentamicin. Additional four, 10-fold or ½ log serialdilutions are made to provide a total of five drug concentrations pluscontrol. Aliquots of 100 μl of these different drug dilutions are addedto the appropriate microtiter wells already containing 100 μl of medium,resulting in the required final drug concentrations.

Following drug addition, the plates are incubated for an additional 48 hat 37° C., 5% CO₂, 95% air, and 100% relative humidity. For adherentcells, the assay is terminated by the addition of cold TCA. Cells arefixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA(final concentration, 10% TCA) and incubated for 60 minutes at 4° C. Thesupernatant is discarded, and the plates are washed five times with tapwater and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4%(w/v) in 1% acetic acid is added to each well, and plates are incubatedfor 10 minutes at room temperature. After staining, unbound dye isremoved by washing five times with 1% acetic acid and the plates are airdried. Bound stain is subsequently solubilized with 10 mM trizma base,and the absorbance is read on an automated plate reader at a wavelengthof 515 nm. For suspension cells, the methodology is the same except thatthe assay is terminated by fixing settled cells at the bottom of thewells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA).Using the seven absorbance measurements [time zero, (Tz), controlgrowth, (C), and test growth in the presence of drug at the fiveconcentration levels (Ti)], the percentage growth is calculated at eachof the drug concentrations levels. Percentage growth inhibition iscalculated as:[(Ti−Tz)/(C−Tz)]×100 for concentrations for which Ti>/=Tz[(Ti−Tz)/Tz]×100 for concentrations for which Ti<Tz

Three dose response parameters are calculated for each experimentalagent. Growth inhibition of 50% (GI₅₀) is calculated from[(Ti−Tz)/(C−Tz)]×100=50, which is the drug concentration resulting in a50% reduction in the net protein increase (as measured by SRB staining)in control cells during the drug incubation. The drug concentrationresulting in total growth inhibition (TGI) is calculated from Ti=Tz. TheLC₅₀ (concentration of drug resulting in a 50% reduction in the measuredprotein at the end of the drug treatment as compared to that at thebeginning) indicating a net loss of cells following treatment iscalculated from [(Ti−Tz)/Tz]×100=−50. Values are calculated for each ofthese three parameters if the level of activity is reached; however, ifthe effect is not reached or is exceeded, the value for that parameteris expressed as greater or less than the maximum or minimumconcentration tested.

The methods described were used to test compounds 70 and 71 on a panelof sixty cell lines under a screening service provided by theDevelopmental Therapeutics Program at the National Institutes of Health.Shown in FIG. 1C are 1, RPMI-8226 leukemia cells; 2; NCI-H322M non-smallcell lung cancer cells; 3, HT29 colon cancer cells; 4, U251 CNS cancercells; 5, SK-MEL-28 melanoma cancer cells; 6, SK-OV-3 ovarian cancercells; 7, RXF 393 renal cancer cells; 8, PC-3 prostate cancer cells; 9,MDA-MB-231 breast cancer cells.

Results of testing compounds 70 and 71 against the full 60 cell linesare shown in the tables below. The testing was done by the NationalCancer Institute Developmental Therapeutics Program. The results shownare in vitro testing results.

EXAMPLE 17

OSU-03012 eliminates intracellular Salmonella typhimurium from infectedmacrophages. The discovery that OSU-03012 induces autophagy in humancancer cells, coupled with the importance of autophagy as a defenseagainst intracellular pathogens, prompted us to test the effect of thisagent on the intracellular survival of S. typhimurium in infected RAW264.7 murine macrophages. Both time- and dose-dependent effects ofOSU-03012 were assessed in intracellular survival (i.e. gentamicinprotection) assays. After 8 h of treatment, OSU-03012 caused dramaticreductions in intracellular bacterial survival with an IC₅₀ of ˜0.2 μMand maximal inhibition at 1 μM (FIG. 3A, B). To determine if OSU-03012had direct bactericidal activity, the effect of OSU-03012 onextracellular bacterial growth was examined in liquid medium (FIG. 3C)and on agar by the disc-diffusion method (not shown). In both systems,the growth of S. typhimurium was unaffected by OSU-03012 atconcentrations of up to 5 μM. These findings indicate that OSU-03012potently eliminates intracellular bacteria from phagocytes and that thisactivity is mediated via an indirect mechanism, presumably througheffects on the host cell.

OSU-03012 eliminates intracellular S. typhimurium by inducing autophagyin infected macrophages. To demonstrate that the elimination ofintracellular bacteria by OSU-03012 treatment is mediated by theinduction of autophagy, infected RAW 264.7 cells were treated with 1 μMOSU-03012 for 8 h in the presence or absence of 3-methyladenine (3MA), aclassical inhibitor of autophagy. FIG. 4A shows that 3MA completelyblocked the ability of OSU-03012 reduce intracellular bacterialsurvival, strongly indicating that autophagy is essential to OSU-03012'sability to eliminate intracellular bacteria. Additional evidence forthis mechanism of action comes from the human breast cancer cell lines,MCF-7 and MDA-MB-231, which differ in their expression levels ofbeclin-1, a tumor suppressor that is critical for the execution ofautophagy (FIG. 5A). Accordingly, the ability of OSU-03012 to induceautophagy in these cell lines, as determined by the intensity ofimmunostaining for endogenous LC3, a marker of mammalian autophagosomes(FIG. 5C), correlates with their respective beclin-1 expression levels.Moreover, intracellular bacterial survival in OSU-03012-treated S.typhimurium-infected breast cancer cells also reflects their respectivebeclin-1 status (FIG. 5B). Rapamycin, an established inducer ofautophagy, is also capable of suppressing survival of S. typhimurium inmurine macrophages (FIG. 4B). However, in comparison to OSU-03012, anearly 100-fold higher concentration of rapamycin is required to matchthe degree of bacterial clearance caused by OSU-03012. Taken together,these data strongly support that OSU-03012 eliminates intracellularpathogens by inducing autophagy in the host cell. Moreover, OSU-03012induces autophagy and bacterial clearance far more potently thanrapamycin and at clinically attainable concentrations.

OSU-03012 eliminates F. novicida from infected macrophages. To obtaindata of direct relevance to the proposed studies, in collaboration withDr. Schlesinger, RAW 264.7 cells were infected with F. novicida andtreated with 1 μM OSU-03012 for 24 h. As shown in FIG. 6, OSU-03012drastically reduced intracellular survival of F. novicida.

To demonstrate that OSU-03012 promotes bacterial clearance inFrancisella-infected macrophages via the induction of autophagy.Rationale. The goal of this is to produce evidence indicating thatOSU-03012, a novel small molecule inducer of autophagy, exhibitsclinical potential as a therapeutic agent against F. tularensisinfection. Using F. novicida initially and then moving to the virulentType A strain of F. tularensis (Schu 4), and human macrophages as thehost cell type, this will extend our studies in S. typhimurium-infectedmurine macrophages to a model of human infection. The proposedexperiments are designed to assess the ability of OSU-03012 to inhibitintramacrophage survival of F. tularensis and to define a role forOSU-03012-induced host cell autophagy in mediating this effect.

Experimental Plan and Methods. Synthesis of OSU-03012. OSU-03012 hasbeen synthesized in large quantities, as depicted in the followingscheme, for in vivo testing in various tumor xenograft models of humancancer and in transgenic animals. OSU-03012 is orally bioavailable withfavorable pharmacokinetic properties, and is currently undergoingpreclinical evaluations under the RAID program at NCI.

Intracellular survival assay to assess Francisella survival inOSU-03012-treated macrophages. Monolayers of J774.1 murine macrophages,THP-1 human macrophages, and human monocyte-derived macrophages (MDM)will be infected with Francisella (MOI of 60:1). After 2 h incubation,extracellular bacteria will be eliminated by addition of gentamicin (50μg/ml; 1 h), followed by washing and treatment with OSU-03012 at variousconcentrations (0.1-5 μM) and for different durations in the presence orabsence of gentamicin (10 μg/ml). After treatments, macrophages will belysed and the lysates serially diluted and plated oncysteine-supplemented Tryptic Soy Agar (TSA) for subsequent enumerationof CFU.

Evaluation of direct bactericidal effects of OSU-03012 on Francisella.To confirm that inhibition of Francisella intracellular survival byOSU-03012 is not a result of direct bactericidal activity against thispathogen, a bacterial growth assay will be performed. Late lag phaseFrancisella will be diluted into antibiotic-free, cysteine-supplementedTryptic Soy Broth and treated with a range of OSU-03012 concentrationsthat encompasses those shown to suppress intracellular survival.Bacterial growth will be monitored by measurements of OD (600 nm) of thebacterial suspensions every 30 min until lag phase is reached.

Evaluation of cytotoxic effects of OSU-03012 on macrophages. The effectof OSU-03012 on the viability of uninfected J774.1 cells, THP-1 cellsand MDMs will be evaluated with the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assayin a 96-well format. Macrophages will be treated with a range ofOSU-03012 concentrations that encompasses those shown to effectivelysuppress intracellular survival. At the end of treatments, the reducedMTT dye will be solubilized and absorbances determined at 570 nm.

Autophagy assays. Pharmacological, immunocytochemical and siRNAapproaches will be used to confirm the induction of autophagy byOSU-03012 in murine and human macrophages, and its role in bacterialkilling.

Effect of autophagy inhibition on OSU-03012-induced suppression ofbacterial survival. Intracellular survival assays will be performed todetermine whether the classical inhibitor of autophagy, 3-methyladenine(3MA), can suppress the ability of OSU-03012 to induce bacterialclearance in macrophages. Likewise, siRNA-mediated knock-down ofbeclin-1, a tumor suppressor critical for the execution of autophagy,will be tested for a similar effect on OSU-03012-induced bacterialkilling. The rescue of Francisella survival in OSU-03012-treatedmacrophages by autophagy inhibition would strongly suggest that anautophagic mechanism mediates OSU-03012 activity. These findings will besupported by the use of cells expressing GFP-labeled LC3, a specificmarker of mammalian autophagosomal membranes. The proportion of cellsexhibiting punctate distribution of GFP-LC3, indicative of autophagy,will be determined in infected macrophages treated with OSU-03012 in thepresence and absence of 3MA- or beclin-1 siRNA-mediated inhibition ofautophagy.

Autolysosome formation in OSU-03012-treated, Francisella-infectedmacrophages. Autophagosome-lysosome fusion and subsequent luminalacidification and lysosomal hydrolysis results in degradation ofautophagocytosed entities. To determine if OSU-03012 induces autophagicmaturation to autolysosome formation, Francisella-infected macrophagesexpressing GFP-LC3 will be treated with OSU-03012 at concentrations thateffectively suppress bacterial survival, and then processed forimmunofluorescence microscopy for the detection of the lysosomal marker,LAMP-1. Enhanced co-localization of GFP-LC3 and LAMP-1-immunopositivityin drug-treated cells would indicate autolysosome formation. Furtherevidence for this phenomenon will be examined in OSU-03012-treatedmacrophages infected with GFP-expressing F. novicida, or Schu 4 strainlabeled with anti-Francisella Ab in which the acidotropic dyeLysoTracker Red (LT) will be used to detect acidifiedbacteria-containing vacuoles. Increases in the proportion ofdrug-treated cells with LT-positive, bacteria-containing vacuoles wouldindicate stimulation of autolysosome formation.

TABLE 5 Results of testing Compound 70 against 60 cancer cell lines.Log10 concentration Panel/Cell Time Mean Optical Densities PercentGrowth Line Zero Ctrl −8.0 −7.0 −6.0 −5.0 −4.0 −8.0 −7.0 −6.0 −5.0 −4.0GI50 TGI LC50 Leukemia CCRF-CEM 0.295 1.099 0.893 0.946 0.523 0.1680.194 74 81 28 −43 −34 3.87E−07 2.49E−06 >1.00E−04  K-562 0.297 1.4671.435 1.022 0.681 0.209 0.247 97 62 33 −30 −17 2.57E−073.34E−06 >1.00E−04  MOLT-4 0.313 1.048 0.964 0.951 0.614 0.180 0.296 8987 41 −42 −6 6.36E−07 3.10E−06 >1.00E−04  RPMI-B226 0.332 0.845 0.7560.701 0.547 0.164 0.229 85 72 42 −51 −31 5.37E−07 2.84E−06 — SR 0.3640.902 0.816 0.796 0.605 0.265 0.363 84 80 45 −27 — 7.11E−074.17E−06 >1.00E−04  Non-Small Cell Lung Cancer A549/ATCC 0.363 1.2791.294 1.149 0.831 0.155 0.252 102 86 51 −57 −31 1.02E−06 2.96E−06 — EKVX0.557 0.860 0.891 0.860 0.786 0.156 0.193 110 100 75 −72 −65 1.49E−063.25E−06 7.08E−06 HOP-62 0.510 1.395 1.310 1.330 1.075 0.155 0.217 90 9364 −70 −57 1.27E−06 3.01E−06 7.13E−06 HOP-92 1.070 1.526 1.509 1.4741.465 0.095 0.267 96 89 87 −91 −75 1.61E−06 3.07E−06 5.87E−06 NCI-H2261.190 1.594 1.593 1.586 1.429 0.505 0.614 100 98 59 −58 −48 1.20E−063.21E−06 — NCI-H23 0.457 1.655 1.672 1.673 1.255 0.097 0.156 101 102 67−79 −66 1.30E−06 2.87E−06 6.33E−06 NCI-H322M 0.626 2.465 1.363 1.5621.420 0.100 0.269 40 51 43 −84 −57 — 2.18E−06 5.40E−06 NCI-H46D 0.3191.843 1.799 1.499 0.830 0.173 0.213 97 77 34 −46 −33 4.21E−072.64E−06 >1.00E−04  NCI-H522 0.766 1.549 1.626 1.718 1.528 0.260 0.486110 122 97 −66 −37 1.95E−06 3.94E−06 — Colon Cancer COLO 205 0.221 1.4121.286 1.252 0.916 0.104 0.239 89 87 58 −53 1 1.19E−06 — — HCT-116 0.1140.708 0.751 0.665 0.452 0.104 0.146 107 93 57 −9 5 1.27E−06 — >1.00E−04 HCT-15 0.279 0.875 0.760 0.599 0.561 0.149 0.278 81 54 47 −47 — 3.65E−073.19E−06 >1.00E−04  HT29 0.215 1.453 1.355 1.299 0.801 0.075 0.159 92 8847 −65 −26 8.58E−07 2.64E−06 — KM12 0.535 2.184 2.151 2.044 1.425 0.1460.264 98 91 54 −73 −51 1.07E−06 2.67E−06 6.62E−06 SW-620 0.187 1.2021.197 1.198 0.736 0.127 0.136 100 100 54 −32 −28 1.12E−064.23E−06 >1.00E−04  CNS Cancer SF-268 0.376 1.142 1.016 0.930 0.7890.252 0.280 84 72 54 −33 −26 1.11E−06 4.16E−06 >1.00E−04  SF-295 0.4211.224 1.173 1.190 0.915 0.225 0.287 94 96 62 −47 −32 1.28E−063.71E−06 >1.00E−04  SNB-19 0.532 1.721 1.562 1.655 1.230 0.219 0.324 8794 59 −59 −39 1.18E−06 3.15E−06 — U251 0.391 1.301 1.298 1.139 0.8100.144 0.320 100 82 46 −63 −18 7.75E−07 2.64E−06 — Melanoma LOX IMVI0.353 1.166 1.204 0.974 0.742 0.176 0.352 105 76 48 −50 — 8.36E−073.07E−06 — MALME-3M 0.666 1.165 1.123 1.128 1.102 0.218 0.290 92 93 87−67 −56 1.74E−06 3.67E−06 7.73E−06 M14 0.315 0.986 0.975 0.974 0.9160.211 0.256 98 98 90 −33 −19 2.10E−06 5.37E−06 >1.00E−04  SK-MEL-2 0.4560.883 0.943 0.956 0.885 0.289 0.330 114 117 100 −37 −28 2.33E−065.40E−06 >1.00E−04  SK-MEL-28 0.530 1.482 1.426 1.320 1.130 0.071 0.21894 83 63 −87 −59 1.22E−06 2.64E−06 5.69E−06 SK-MEL-5 0.549 2.147 1.9951.958 1.659 0.136 0.364 91 88 69 −75 −34 1.36E−06 3.02E−06 — UACC-620.675 1.600 1.649 1.546 1.407 0.137 0.364 105 94 79 −80 −46 1.53E−063.15E−06 — Ovarian Cancer IGROV1 0.462 1.060 1.044 1.103 0.902 0.2350.302 97 107 74 −49 −35 1.56E−06 3.97E−06 >1.00E−04  OVCAR-3 0.460 0.9290.879 0.849 0.816 0.140 0.301 89 83 76 −70 −35 1.51E−06 3.32E−06 —OVCAR-4 0.530 1.121 1.114 1.073 1.086 0.093 0.172 99 92 94 −82 −681.78E−06 3.41E−06 6.55E−06 OVCAR-5 0.512 1.507 1.480 1.540 1.412 0.0870.149 97 103 90 −83 −71 1.71E−06 3.32E−06 6.45E−06 OVCAR-8 0.400 1.1011.078 1.114 0.755 0.269 0.306 97 102 51 −33 −24 1.02E−064.05E−06 >1.00E−04  SK-OV-3 0.399 1.623 1.514 1.597 1.262 0.083 0.302 9198 71 −79 −24 1.37E−06 2.96E−06 — Renal cancer 78G-0 0.652 1.958 2.0121.897 1.440 0.181 0.299 104 95 60 −72 −54 1.20E−06 2.85E−06 6.79E−06A498 0.945 2.261 2.230 2.598 2.201 0.660 0.433 98 126 95 −30 −542.30E−06 5.75E−06 6.69E−05 ACHN 0.374 1.026 0.985 1.037 0.829 0.1500.313 94 102 70 −60 −16 1.42E−06 3.45E−06 — CAKI-1 0.597 1.862 1.6921.802 1.126 0.147 0.335 87 95 42 −75 −44 7.03E−07 2.27E−06 — RXP 3930.403 0.801 0.766 0.700 0.628 0.177 0.441 91 75 56 −56 10 1.14E−06 — —SN12C 0.584 1.233 1.209 1.194 1.093 0.203 0.308 96 94 78 −65 −471.58E−06 3.51E−06 — TK-10 0.672 1.496 1.512 1.492 1.304 0.138 0.200 102100 77 −80 −70 1.48E−06 3.10E−06 6.47E−06 UO-31 0.443 1.782 1.572 1.5631.356 0.161 0.285 84 84 68 −64 −36 1.37E−06 3.29E−06 — Prostate CancerPC-3 0.242 0.996 0.910 0.763 0.593 0.055 0.108 89 69 47 −77 −55 7.05E−072.38E−06 6.02E−06 DU-145 0.327 0.961 0.993 1.018 0.895 0.078 0.132 105109 90 −76 −60 1.73E−06 3.47E−06 6.95E−06 Breast cancer MCF7 0.452 1.6721.566 1.541 0.982 0.196 0.255 91 89 43 −57 −44 7.18E−07 2.72E−06 —NCI/ADR-RES 0.556 1.904 1.883 1.878 1.217 0.229 0.212 98 98 49 −59 −629.54E−07 2.85E−06 8.28E−06 MDA-MB- 0.642 0.988 0.908 0.889 0.843 0.1730.306 100 71 58 −73 −52 1.15E−06 2.77E−06 6.67E−06 231/ATCC HS 57BT0.534 1.186 1.221 1.179 1.110 0.351 0.333 105 99 88 −34 −38 2.05E−065.25E−06 >1.00E−04  MDA-MB-435 0.404 1.469 1.502 1.447 1.126 0.127 0.347103 98 68 −69 −14 1.35E−06 3.14E−06 — BT-549 0.492 0.957 0.912 0.8910.785 0.143 0.195 90 86 63 −71 −60 1.25E−06 2.95E−06 6.98E−06 T-47D0.423 1.019 0.921 1.071 0.935 0.205 0.238 84 109 86 −52 −44 1.82E−064.21E−06 —

TABLE 6 Results of testing Compound 71 against 60 cancer cell lines.Log10 Concentration Panel/Cell Time Mean Optical Densities PercentGrowth Line Zero Ctrl −8.0 −7.0 −6.0 −5.0 −4.0 −8.0 −7.0 −6.0 −5.0 −4.0GI50 TGI LC50 Leukemia CCRF-CEM 0.295 0.949 0.808 0.811 0.718 0.0020.140 78 79 65 −99 −53 1.23E−06 2.48E−06 5.00E−06 K-562 0.297 1.4011.338 1.283 0.438 0.126 0.119 94 89 13 −58 −60 3.26E−07 1.52E−067.77E−06 MOLT-4 0.313 0.957 0.833 0.736 0.461 0.143 0.143 81 66 23 −54−54 2.33E−07 1.98E−06 8.75E−06 RPMI-8226 0.332 0.682 0.536 0.559 0.5130.016 0.176 58 65 52 −95 −47 1.03E−06 2.25E−06 — SR 0.364 0.888 0.6880.583 0.254 0.234 0.287 62 42 −30 −36 −21 3.86E−08 3.79E−07 >1.00E−04 Non-Small Cell Lung Cancer A549/ATCC 0.363 1.175 1.140 1.145 1.121−0.032 0.135 96 96 93 −100 −63 1.68E−06 3.04E−06 5.51E−06 EKVX 0.5570.841 0.713 0.737 0.711 −0.008 0.145 55 63 54 −100 −74 1.06E−06 2.24E−064.74E−06 HOP-62 0.510 1.288 1.285 1.267 1.255 0.011 0.329 100 97 96 −98−36 1.72E−06 3.12E−06 — HOP-92 1.070 1.647 1.567 1.541 1.361 0.089 0.76786 82 50 −92 −28 1.01E−06 2.26E−06 — NCI-H23 0.457 1.669 1.585 1.5151.679 −0.041 0.151 93 87 101 −100 −67 1.79E−06 3.18E−06 5.64E−06NCI-H322H 0.626 1.181 0.988 1.049 1.031 −0.052 0.257 65 76 73 −100 −591.36E−06 2.64E−06 5.14E−06 NCI-H460 0.319 1.792 1.654 1.658 1.650 0.0110.257 91 91 90 −97 −20 1.64E−06 3.04E−06 — NCI-H522 0.766 1.671 1.5091.558 1.471 0.037 0.439 82 87 78 −95 −43 1.45E−06 2.82E−06 — ColonCancer COLO 205 0.221 1.196 1.146 1.028 0.930 0.009 0.151 95 83 73 −96−32 1.36E−06 2.69E−06 — HCT-116 0.114 0.714 0.607 0.633 0.525 −0.0220.058 82 86 68 −100 −49 1.29E−06 2.55E−06 — HCT-15 0.279 0.893 0.6690.779 0.797 −0.003 0.082 64 81 84 −100 −71 1.54E−06 2.87E−06 5.36E−06HT29 0.215 1.333 1.276 1.238 1.136 −0.054 0.003 95 91 82 −100 −991.50E−06 2.83E−06 5.32E−06 KM12 0.535 1.957 1.867 1.832 1.777 0.0130.307 94 91 87 −98 −43 1.59E−06 2.97E−06 — SW-620 0.187 1.073 0.9230.993 1.060 −0.048 0.015 83 91 98 −100 −92 1.75E−06 3.13E−06 5.60E−06CNS Cancer SF-268 0.376 1.016 0.893 0.939 0.886 0.148 0.278 81 88 80 −61−26 1.63E−06 3.69E−06 — SF-295 0.421 1.107 1.033 0.931 0.997 −0.0250.195 89 74 84 −100 −54 1.53E−06 2.86E−06 5.35E−06 SNB-19 0.532 1.4831.385 1.385 1.351 0.063 0.317 90 90 86 −88 −40 1.61E−06 3.12E−06 — U2510.391 1.156 1.125 1.059 1.123 0.026 0.484 96 87 96 −93 12 1.74E−06 — —Melanoma LOX IMVI 0.353 1.164 0.948 1.033 0.935 0.008 0.165 73 84 72 −98−53 1.34E−06 2.65E−06 5.22E−06 MALME-3M 0.666 0.984 0.832 0.838 0.8260.089 0.333 52 54 50 −87 −50 1.01E−06 2.33E−06 1.00E−04 M14 0.315 0.9350.772 0.823 0.816 −0.004 0.210 74 82 81 −100 −33 1.48E−06 2.80E−06 —SK-MEL-1 0.456 0.830 0.775 0.819 0.780 −0.010 0.324 85 97 87 −100 −291.57E−06 2.91E−06 — SK-MEL-28 0.530 1.508 1.439 1.408 1.367 −0.002 0.24893 90 86 −100 −53 1.56E−06 2.89E−06 5.38E−06 SK-MEL-5 0.549 1.640 1.6111.762 1.812 0.143 0.363 97 111 116 −74 −34 2.22E−06 4.07E−06 — UACC-620.675 1.477 1.323 1.338 1.301 −0.041 0.272 81 83 78 −100 −60 1.44E−062.74E−06 5.24E−06 Ovarian Cancer IGROV1 0.462 1.091 0.997 0.983 0.806−0.039 0.216 85 83 55 −100 −53 1.07E−06 2.26E−06 4.75E−06 OVCAR-3 0.4600.868 0.833 0.792 0.770 −0.043 0.131 91 81 76 −100 −72 1.40E−06 2.70E−065.20E−06 OVCAR-4 0.530 1.123 1.011 0.999 0.945 −0.001 0.179 81 79 70−100 −66 1.31E−06 2.58E−06 5.08E−06 OVCAR-5 0.512 1.592 1.538 1.4371.559 −0.033 0.474 95 86 97 −100 −8 1.73E−06 3.11E−06 — OVCAR-8 0.4000.968 0.923 0.946 0.934 −0.001 0.126 92 96 94 −100 −69 1.69E−06 3.05E−065.52E−06 SK-OV-3 0.399 1.327 1.261 1.245 1.093 0.062 0.278 93 91 75 −85−30 1.43E−06 2.94E−06 — Renal Cancer 786-0 0.652 1.912 1.862 1.868 1.6630.046 0.458 96 96 80 −93 −30 1.49E−06 2.91E−06 — A498 0.945 2.207 2.4132.087 1.869 1.903 0.540 116 91 73 76 −43 1.65E−05 4.35E−05 >1.00E−04 ACHN 0.374 1.026 0.898 0.902 0.964 0.034 0.291 80 81 90 −91 −22 1.67E−063.15E−06 — CAKI-1 0.597 1.767 1.735 1.642 1.804 −0.067 0.315 97 89 103−100 −47 1.83E−06 3.22E−06 — RXF 393 0.403 0.829 0.582 0.629 0.629 0.1560.385 42 53 53 −61 −4 — 2.91E−06 — SN12C 0.584 1.115 1.050 0.998 1.045−0.014 0.224 88 78 87 −100 −62 1.57E−06 2.91E−06 5.40E−06 TK-10 0.6721.134 1.032 1.087 0.998 −0.021 0.245 78 90 71 −100 −64 1.32E−06 2.59E−065.09E−06 UO-31 0.443 1.467 1.375 1.407 1.332 −0.030 0.288 91 94 87 −100−35 1.57E−06 2.92E−06 — Prostate Cancer PC-3 0.242 1.068 0.862 0.8460.857 −0.022 0.179 75 73 75 −100 −26 1.38E−06 2.67E−06 — DU-145 0.3270.835 0.694 0.767 0.760 −0.074 −0.019 72 87 85 −100 −100 1.55E−062.89E−06 5.37E−06 Breast Cancer MCF7 0.452 1.540 1.467 1.340 1.507 0.0730.229 93 82 97 −84 −49 1.82E−06 3.44E−06 — NCI/ADR-RES 0.556 1.942 1.9201.882 1.874 0.490 0.294 98 96 95 −12 −47 2.64E−06 7.73E−06 >1.00E−04 MDA-MB- 0.642 0.884 0.756 0.830 0.709 0.015 0.389 47 78 27 −98 −39 —1.66E−06 — 231/ATCC HS 578T 0.534 1.167 1.099 1.165 1.127 0.168 0.333 89100 94 −69 −38 1.86E−06 3.78E−06 — MDA-MB-435 0.404 1.498 1.376 1.3821.444 −0.073 0.295 89 89 95 −100 −27 1.70E−06 3.07E−06 — T-47D 0.4230.750 0.541 0.578 0.529 0.100 0.198 36 47 32 −76 −53 <1.00E−08  1.98E−065.72E−06

The examples described herein are meant to be illustrative of thesynthesis and applications of the compounds described. The examples arenot meant to limit the scope of the invention described herein.

TABLE 7 Treatment groups & mouse numbers: Total = 250 mice TreatmentsGentamicin^(B) OSU-03012 (mg/kg; p.o., QD)^(A) Regimen Vehicle (100 μg,i.n.) 50 100 200 A^(C) 20 20 20 20 20 B^(C) 20 20 20 20 20 C 10 10 10 1010 ^(A)These doses of OSU-03012 are based on our experience with thisagent in mouse models of cancers in which these doses causeddose-dependent suppression of tumor growth, and attained average plasmalevels of 8-10 μM. This plasma level is higher than that shown in ourpreliminary studies to induce bacterial clearance and autophagy inmurine macrophages and human breast cancer cells. ^(B)Experiments foreach Regimen will be conducted separately; thus each requires its owngentamicin treatment group. ^(C)Mouse numbers for each group in RegimensA and B include 10 mice to be sacrificed at 24 h post-infection forassessment of bacterial load in target organs.

1. A compound of Formula I

wherein X is —CF₃, Ar is

and R is selected from

R′ is is SO₂CH₂CH₂NH₂, or SO₂NH₂ or an amino acid attached through theα-carboxyl group selected from the group consisting of L-Lys, D-Lys,β-Ala, L-Lue, L-Ile, Phe, Asn, Glu and Gyl, and R″ is methyl, ethyl,allyl, CH₂CH₂OH, CH₂CN, CH₂CH₂CN, CH₂CONH₂,

or pharmaceutically acceptable salts thereof.
 2. The compound of claim 1wherein the compound has the following Formula VI

or pharmaceutically acceptable salts thereof.
 3. The compound of claim 1wherein the compound has the following Formula VII

or pharmaceutically acceptable salts thereof.
 4. The compound of claim 1wherein the compound has the following Formula XIV XIV

N4NBS R″ =

MW 657.66 N4BPS R″ =

MW 688.72 N4BBS R″ =

MW 691.56 N4MBS R″ =

MW 626.69 N4ME R″ = Me MW 486.53 N4E R″ = Et MW 500.56 N4ALL R″ = AllylMW 512.57 N4HE R″ = CH2CH2OH MW 516.56 N4ACN R″ = CH2CN MW 511.54 N4PCNR″ = CH2CH2CN MW 525.57 N4ETFM R″ = CH2CH2CF3 MW 568.56 N4AA R″ =CH2CONH2

or pharmaceutically acceptable salts thereof.
 5. A compound of thefollowing Formula IX

or pharmaceutically acceptable salts thereof.
 6. A method of inducingapoptosis in cancer cells selected from the group consisting ofleukemia, non-small cell lung cancer, colon cancer, central nervoussystem cancer, melanoma, ovarian cancer, renal cancer, prostate cancer,and breast cancer cells, the method comprising the step of contacting atherapeutically effective amount of a compound of Formula I

wherein X is —CF₃, Ar is

and R is selected from

 where R′ is is SO₂CH₂CH₂NH₂, or SO₂NH₂ or an amino acid attachedthrough the α-carboxyl group selected from the group consisting ofL-Lys, D-Lys, β-Ala, L-Lue, L-Ile, Phe, Asn, Glu and Gyl, and R″ ismethyl, ethyl, allyl, CH₂CH₂OH, CH₂CN, CH₂CH₂CN, CH₂CONH₂,

or pharmaceutically acceptable salts thereof, with the rapidlyproliferating cells.
 7. A method for treating, inhibiting , or delayingthe onset of cancer, wherein the cancer is selected from the groupconsisting of leukemia, non-small cell lung cancer, colon cancer,central nervous system cancer, melanoma, ovarian cancer, renal cancer,prostate cancer, and breast cancer, in a subject in need of suchtreatment, the method comprising administering a therapeuticallyeffective amount of a compound of Formula I:

wherein X is —CF₃, Ar is

and R is selected from

 where R′ is is SO₂CH₂CH₂NH₂, or SO₂NH₂ or an amino acid attachedthrough the α-carboxyl group selected from the group consisting ofL-Lys, D-Lys, β-Ala, L-Lue, L-Ile, Phe, Asn, Glu and Gyl, and R″ ismethyl, ethyl, allyl, CH₂CH₂OH, CH₂CN, CH₂CH₂CN, CH₂CONH₂,

or pharmaceutically acceptable salts thereof, to the subject in need ofsuch treatment.
 8. The method of claim 7 wherein the subject is a human.9. A method of inducing autophagy in cells infected by an intracellularbacteria comprising administering to a subject diagnosed with a diseasecaused by the bacteria a therapeutically effective amount of at leastone compound selected from Formula I, VI, VII, IX, and XIV.
 10. Themethod according to claim 9, wherein the subject is an animal.
 11. Themethod according to claim 10, wherein the subject is a human.
 12. Themethod according to claim 9, wherein the bacteria is chosen fromMycobacterium tuberculosis, Francisella tularensis, Streptococcuspyogenes, Rickettsiae spp., and Salmonella typhimurium.
 13. A method ofreducing the release of bacterial endotoxins in a subject comprisingadministering to a subject that has undergone antibiotic treatment atherapeutically effective amount of at least one compound selected fromFormula I, VI, VII, IX, and XIV.
 14. The method according to claim 13,wherein the subject is an animal.
 15. The method according to claim 14,wherein the subject is a human.